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Neuron, Volume 79

Supplemental Information

SAD Kinases Sculpt Axonal Arbors of Sensory

Neurons through Long- and Short-Term

Responses to Neurotrophin Signals Brendan N. Lilley, Y. Albert Pan, and Joshua R. Sanes  

SUPPLEMENTAL INFORMATION INVENTORY:

Figure S1: Deletion of LKB1 in neural progenitors using Nestin-cre causes defects in cortical development. Related to Figure 1.

Figure S1 demonstrates that deletion of LKB1 in neural progenitors produces defects in cortical development seen previously with Emx1-cre mediated deletion (Barnes et al., 2007), although these same animals do not exhibit defects in axon development elsewhere in the nervous system (Figure 1).

Figure S2: Conditional knockout of SAD and LKB1 kinases with Isl1-cre. Related to Figure 2.

Figure S2 demonstrates the design and utility of mouse lines used (the SAD-A and LKB1 conditional and Isl1-cre lines), and provides additional supporting data regarding the gross phenotype and IaPSN axon arborization phenotype of SADIsl1-cre mutants.

Figure S3: SAD kinases are required in NT-3 dependent sensory neurons for central, but not peripheral axon differentiation. Related for Figure 3.

Figure S3 includes the demonstration of: axon arborization defects in SAD-deficient whisker afferents in the SpVi, cell autonomous requirements for SADs in sensory axon arborization and non-requirement for SADs in peripheral axon differentiation. These results extend findings reported in Figure 3.

Figure S4: SAD kinases are not required for TrkC expression or retrograde NT-3 signaling in IaPSNs. Related to Figure 4.

Figure S4 provides additional data showing that SADs are not required for expression of the NT-3 receptor TrkC or for retrograde NT-3 signaling (extending findings in Figure 4).

Figure S5: MAP3K7/TAK1 activates SADs in vitro, but is not a relevant activator in vivo. Related to Figure 6.

Figure S5 provides data in support of Figure 6 and demonstrates that TAK1/MAP3K7 is a novel SAD ALT kinase that is expressed in DRG neurons, but is not required for SAD function in axon arborization in vivo.

Figure S6: Regulation of SAD kinase activation by inhibitory phosphorylation in the C-terminal domain. Related to Figures 6 and 7.

Figure S6 provides supporting data for Figures 6 and 7 by showing the amino acid sequence of the SAD-A and SAD-B CTDs and sites of phosphorylation, further evidence for the role of CTD phosphorylation in regulating SAD ALT phosphorylation (Figure 6) and the Ca2+ dependence of SAD-A CTD dephosphorylation (Figure 7).

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Figure S1: Deletion of LKB1 in neural progenitors using Nestin-cre causes defects in cortical

development. Related to Figure 1. Immunohistochemical analysis of E18.5 cortex with Nissl (A,B), and

Tau1 (A’,B’) shows loss of LKB1 results in dramatic thinning of the cortical wall, reduced Tau1-positive

axon tracts in the intermediate zone (IZ), increased Tau1 staining in the cortical plate, and increased

numbers of pyknotic nuclei. Scale bar, 50 m.

LKB1Nestin-creControl

Tau1Nissl

Lilley, Pan and SanesFigure S1

A A’ B B’

Tau1Nissl

MZ

CP

IZ

SVZ

MZ

CP

IZ

SVZ

LV

Lilley, Pan and Sanes

Figure S2

SAD-A genomic

Targeting vector

Floxed allele

+FLP

+Cre

1

XbaI

XbaI

KpnI

ClaI

2x NotI

KpnI

Eco

RI

1 PGK-NEOR

1 PGK-NEOR

1

1 Kb

PCR

A Lumbar SC

Af/f;Cre+Af/+

SAD-A

SAD-B

actin

B

STOPRosa-CAG GFP -Cre=

Rosa-CAG GFP +Cre=GFP

X Isl1 IRES-Cre-pA

C

Control SADIsl1cre

G H

E13-TG and Brainstem E13-DRG,

Spinal cord

GFP - Isl1/2

GFP - Isl1/2

D

E

Cervical Thoracic Medial Lumbar

P0 Spinal Cord - PV

Control

SADIsl1cre

I

J

K

L

M

N

Isl1Cre+

LKB1

TUJ1

LKB1fl/+

LKB1fl/+

LKB1fl/fl

F

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Figure S2: Conditional knockout of SAD and LKB1 kinases with Isl1-cre. Related to Figure 2. (A)

Strategy for targeting the SAD-A locus to generate the conditional allele. (B) Immunoblotting for SAD-A

and SAD-B from lysates of spinal cord showing loss of SAD-A protein in conditional knockouts. (C)

Schematic of lines used for analysis of Isl1-cre activity. (D,E) Immunohistochemical analysis of tissue

from E13.5 Isl1-cre; RC::Epe animals using antibodies against GFP and Isl1/2. GFP signal is prominent

in trigeminal and dorsal root ganglion and axons of sensory neurons entering the CNS. Motor neurons

and dI3 interneurons are also GFP+ in spinal cord. (F) Immunoblotting of lysates from cultured DRG

neurons shows loss of LKB1 protein in conditional knockout neurons relative to controls. Residual LKB1

protein likely comes from contaminating glia and fibroblasts. (G-H) Neonatal control animals have large

milk spots, but SADIsl1-cre mutants do not. (I-N) Parvalbumin immunohistochemistry of different levels of

P0 spinal cord showing defects in SADIsl1-cre mutants at all levels. Scale bars, 100 m.

Lilley, Pan and SanesFigure S3

Control SADIsl1cre

P0 BSTC - NF-H

C DControl SADChAT-cre

P8 Lumbar Spinal Cord - Nissl/PV

G H

Control

SADIsl1cre

PV SytII Merge

E17

.5 -

Tric

eps

K’ K’’

L L’ L’’

K

Control SADIsl1cre

E13.5 Lumbar Spinal Cord - Isl1/2

E F

IP6-BSTC-SpVi

I’

GFP - TOPRO - VGLUT1

GFP

P6-Dorsal Column NucleiJ

J’

GFP

GFP - Nissl - VGLUT1

N

Control SADIsl1-cre

E15

DR

G -

PV

M N

Control SADIsl1cre

P0

SpV

i - D

iIAA B

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Figure S3: SAD kinases are required in NT-3 dependent sensory neurons for central, but not

peripheral axon differentiation. Related for Figure 3. (A,B) Transganglionic DiI labeling and

visualization of central axon arbors in BSTC subnucleus interpolaris (SpVi), shows SADs are required for

terminal axon arbor formation in the BSTC. (C,D) Neurofilament immunohistochemistry of P0 BSTC,

subnucleus interpolaris (SpVi) shows structure of target region of whisker afferents to be similar between

control and SADIsl1-cre animals. (E,F) Isl1+ dI3 interneurons survive and migrate to the medial spinal cord

in E13.5 SADIsl1-cre mutants. (G,H) Deletion of SADs in motor neuron targets of IaPSNs with ChAT-cre

does not affect axon innervation of the ventral horn. (I-J’) Isl1-cre reporter analysis at P6 in brainstem

targets of IaPSN (DCN, I,I’’) and whisker afferents (SpVi, J,J’) shows that only incoming axons express

Cre reporter; no cells in any of the target regions expressed Isl-cre. (K-L’’) PV and SytII

immunohistochemistry of E17.5 limb muscles shows differentiation of annulospiral endings on intrafusal

muscle fibers. (M,N) Pseudounipolar morphology of PV+ DRG neurons develops in SADIsl1-cre mutants by

E15.5. Scale bars A,B, M,N: 50 m, C-L’’: 100 m.

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Figure S4: SAD kinases are not required for TrkC expression or retrograde NT-3 signaling in

IaPSNs. Related to Figure 4. (A-B’’) PV and TrkC staining of P0 DRG shows expression of TrkC in PV+

cell bodies. (C-H) ER81 immunohistochemistry beginning at E13.5 shows that SADs are not required for

retrograde NT-3 signaling or for development of the NT-3 phenotype. Scale bars, 50 m.

Lilley, Pan and SanesFigure S4

DRG - ER81

Control SADIsl1-cre

E15

.5E

14

.5E

13

.5

Control SADIsl1-cre

PV

Trk

CM

erg

e

P0 DRG

C

E

G

D

F

H

A

A’

A’’

B

B’

B’’

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Figure S5: MAP3K7/TAK1 activates SADs in vitro, but is not a relevant activator in vivo. Related

to Figure 6. (A) HeLa cells were transfected with the indicated LKB1 or B-RAF constructs and SAD-AWT.

Serum starved cells or starved cells treated with serum for 15 min were analyzed by immunoblotting. (B)

HeLa cells expressing LKB1 or TAK1/TAB1 constructs shows that TAK1 activation induces SAD ALT

phosphorylation. (C) Kinase assays with purified SAD-A or SAD-B and LKB1 or TAK1/TAB1 were

incubated for indicated times followed by immunoblotting with the antibodies indicated. LKB1 and

TAK1/TAB1 potently phosphorylate SAD proteins on the ALT. (D) TAK1 and TAB1 are expressed in

DRG neuron cultures and whole DRG at different ages. (E-G) PV immunohistochemistry of P0 lumbar

spinal cord shows that neither TAK1 nor LKB1 are required for ventral horn innervation by IaPSNs. Scale

bar,50µm.

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Figure S6: Regulation of SAD kinase activation by inhibitory phosphorylation in the C-terminal

domain. Related to Figures 6 and 7. (A) Amino acid sequence of the SAD-A and SAD-B CTDs.

Identified sites of phosphorylation in mouse brain (Phosphomouse, Huttlin et al., 2010) that were mutated

to Ala are in red (SAD-A only), proline directed sites are underlined, the PXX[S/T]P repeat is boxed and

the D-box is in blue. (B) 293T cells were transfected with the indicated SAD-A and CDK5 constructs.

Active CDK5 completely suppresses SAD ALT phosphorylation of SAD-AWT, SAD-A13A and SAD-A5A but

has a less of an effect on SAD-A18A. (C) Chelation of extracellular Ca2+ blocks A23187 induced SAD-A

CTD dephosphorylation in HeLa cells.

Lilley, Pan and SanesFigure S6

SAD-A aa 421-654LSTSPLSSPR VTPHPSPRGS PLPTPKGTPV HTPKESPAGT PNPTPPSSPS VGGVPWRTRL NSIKNSFLGS PRFHRRKLQV PTPEEMSNLT PESSPELAKKSWFGNFINLE KEEQIFVVIK DKPLSSIKAD IVHAFLSIPS LSHSVISQTSFRAEYKATGG PAVFQKPVKF QVDITYTEGG EAQKENGIYS VTFTLLSGPSRRFKRVVETI QAQLLSTHDQ PSAQHLSGII PKS

SAD-B aa 441-776SPLSSPRSPV FSFSPEPGAG DEARGGGSPT SKTQTLPSRG PRGGGAGEQP PPPSARSTPL PGPPGSPRSS GGTPLHSPLH TPRASPTGTP GTTPPPSPGGGVGGAAWRSR LNSIRNSFLG SPRFHRRKMQ VPTAEEMSSL TPESSPELAKRSWFGNFISL DKEEQIFLVL KDKPLSSIKA DIVHAFLSIP SLSHSVLSQTSFRAEYKASG GPSVFQKPVR FQVDISSSEG PEPSPRRDGS SGGGIYSVTFTLISGPSRRF KRVVETIQAQ LLSTHDQPSV QALADEKNGA QTRPAG TPPRSLQPPPGRSD PDLSSSPRRG PPKDKKLLAT NGTPLP

+- +-A23187 K252a

+--

Tubulin

GFP

SAD-A

pERK1/2

EGTA

A

LKB1

SAD-A

pSAD (ALT)

p35

CDK5-WT++

++ CDK5-KD

++

++

SAD-AWT 13A 5A 18A CB

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SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Animals

Animals were used in accordance with NIH guidelines and protocols approved by Institutional Animal

Use and Care Committee at Harvard University. SAD-A and SAD-B null animals have been described

(Kishi et al., 2005). The Isl1-cre line (Isl1tm1(cre)Tmj, Srinivas et al., 2002) was provided by Tom Jessell

(Columbia University). The Chat-cre (Chattm1(cre)Lowl, Rossi et al., 2011) and Bax null (Baxtm1Sjk, Knudson

et al., 1995) lines were obtained from Jackson Laboratories. Nestin-Cre, Tg(Nes-cre)1Kln (Tronche et al.,

1999), was generated by Rudiger Klein (Max Planck Institute of Neurobiology) and was provided by

Andrew McMahon (Harvard University). The LKB1 (Stk11tm1.1Rdp, Bardeesy et al., 2002) and TAK1

(Map3k7tm1Mds, Liu et al., 2006) conditional lines were provided by Ron DePinho (Dana Farber Cancer

Research Institute) and Michael Schneider (Imperial College London), respectively. The RC::Epe cre

reporter line, Gt(ROSA)26Sortm6Dym (Purcell et al., 2009) was provided by Susan Dymecki (Harvard

Medical School). The SAD-A floxed allele was generated by inserting loxP sites around exon 1 of the

SAD-A gene and a FRT-pgk-NEO-FRT cassette. The targeting construct was linearized and

electroporated into R1 ES cells. Two clones underwent homologous recombination based on screening

by PCR and were used for blastocyst injection. High percentage chimeras transmitting the SAD-A floxed

allele were bred to animals expressing FLP recombinase from the beta-actin promoter (Rodriguez et al.,

2000) to remove the PGK-NEO cassette. Primers used for genotyping were:

KOSDACF-F (AGTATGTGGGGCCCTACCGGCTGGAGAAGA) and

KOSDACF-R (ATGTCCTTGGGCTGCACCCGCCCTCCTGC)

Animals were maintained on a mixed genetic background. Timed pregnant females were obtained by

daily examination for vaginal plug, noon of the day of vaginal plug was denoted E0.5. Timed pregnant

CD-1 females were obtained from Charles River Laboratories.

Immunohistochemistry and DiI labeling

Embryos younger than E15.5 were immersion fixed in 4% paraformaldehyde in 0.1 M Phosphate buffer

pH 7.2 (PB) on ice for 4 hours. Older embryos and neonates were transcardially perfused with Ringer

followed by fixative. After thorough washing in 0.1M PB, tissue was incubated overnight at 4°C in 30%

w/v sucrose in 0.1M PB then was embedded in OCT medium (TissueTek) and frozen. Sections (12-20

microns) were cut on a cryostat, mounted on glass slides and air-dried. After rehydration in TBS, tissue

sections were incubated in blocking buffer (TBS with 10% Normal goat serum, 0.3% Triton X-100) for 2

hours, and were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. Slides

were washed in TBS and incubated for 2 hours at room temperature secondary antibodies diluted in

blocking buffer. Following additional washes, sections were incubated with Neurotrace 435 (Invitrogen)

and mounted with Vectashield (Vector Laboratories).

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The following antibodies (and their sources) were used for immunohistochemistry: anti-Islet1/2 (40.D5),

mouse mAb TROMA-I, anti-SytII mAb ZNP1 (Developmental Studies Hybridoma Bank, developed under

the auspices of NICHD and maintained by the University of Iowa); mouse mAb Tau1, rabbit anti-CGRP,

chicken anti-GFP, guinea-pig anti-VGLUT1 (Millipore); rabbit anti-TrkC, rabbit mAb anti-BRSK2 (found to

be cross-reactive with Brsk1) and activated Caspase-3 (Cell Signaling Technologies); anti-NF-H mAb

SMI312, mouse mAb TUJ1 (Covance); mouse mAb anti-MAP2 (Sigma); rabbit and goat anti-parvalbumin

(Swant); rabbit anti-TrkA (kind gift from Louis Reichardt, UCSF); rabbit anti-ER81 (kind gift from Tom

Jessell, Columbia University). Dye-conjugated secondary antibodies were from Invitrogen and Jackson

Immunoresearch.

For DiI labeling experiments, E15-P0 animals were transcardially perfused with 4% PFA and the spinal

column and head were removed and postfixed for 24 hours. For DRG labeling, the ventral spinal cord

surrounding either the L1 or T12 DRG was exposed by removing the overlying vertebral column. A small

crystal of DiI (~200 um, Invitrogen) was placed into the DRG and the tissue was immersed in fixative at

37°C for 4-6 weeks. Transganglionic labeling of whisker afferents was performed by removing the B2 or

C2 whisker and inserting a DiI crystal into the follicle. The head was placed in fixative at 37°C for 6-8

weeks. Labelling was monitored periodically by examination with a fluorescence dissection microscope.

After labeling, the spinal cord or brainstem was dissected out and was embedded in 3% agarose.

Transverse spinal cord and horizontal brainstem sections were cut at 100 um on a vibratome and were

mounted on glass slides and coverslipped with PB. Samples were imaged within 24 hours of sectioning

to reduce dye spread within the sample. All imaging was done on an Olympus FV1000 confocal

microscope. ImageJ was used to analyze confocal stacks and generate maximum intensity projections.

Images were processed in Adobe Photoshop.

Plasmids

SAD-A expression plasmids were constructed by inserting the mouse SAD-A cDNA (alpha isoform) into

the BamHI site of pCDNA-FLAG/HA (obtained from William Sellers through Addgene, plasmid #10792).

Site directed mutagenesis was used to introduce point mutations and all constructs were verified by

sequencing. To make GFP::SAD-A constructs, the SAD-A ORF was amplified by PCR and inserted into

pENTR-1A DUAL (Invitrogen). Gateway cloning was used to insert the SAD-A ORF into pCS2-EGFP-

DEST (Obtained from Nathan Lawson through Addgene, #13071). The GFP::SAD-A ORF was then

subcloned into pFUGW (Addgene #14883) to produce lentivirus. For bacterial expression of SAD-A and

SAD-B, the complete ORF was cloned into pGEX6P-1 (GE Healthcare). Constructs encoding GFP-

4Rtau, rat TrkC and LKB1 have been described (Kishi et al. 2005; Apel et al., 1996; Barnes et al., 2007).

HA-CDK5-WT and -DN (Addgene #s 1872, 1873) and p35 (Addgene # 1347) expression plasmids were

obtained from Sander Van den Heuvel (Utrecht University) and Li-Huei Tsai (Picower Institute, MIT)

through Addgene. TAK1 and TAB1 constructs were kind gifts from Marian Carlson (Columbia University).

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B-RAF expression constructs were kind gifts of Hans Widlund (Dana Farber Cancer Research Institute).

The B-RAF V600E lentiviral construct was made by subcloning the B-RAF V600E ORF into pENTR1A

followed by transfer into pLenti-UbC-V5-DEST (Invitrogen).

DRG neuron cultures

Timed pregnant females were sacrificed at E12 or E13 and embryos were dissected in HBSS with 10mM

HEPES buffer and 20mM D-glucose. For dissociated cultures, DRGs were removed from the spinal

cords, pooled in an eppendorf tube, and treated with collagenase (Worthington) followed by 0.05%

Trypsin (Invitrogen) for 10 minutes each at 37°C. Both proteases were dissolved in HBSS with 10mM

HEPES buffer and 20mM D-glucose. The mixture was triturated until a single cell suspension was

achieved. The cells were then washed twice with NB+ (Neurobasal with 1X B27, 1X N2, 1mM Glutamax-

I, 10 M Uridine and 10 M 5’-fluorodeoxyuridine) media containing soybean trypsin inhibitor

(Worthington) to neutralize trypsin. Cells were plated at high density (DRGs of 1 embryo per 4 wells) in

48 well dishes coated with 0.1 mg/ml poly-L lysine (Sigma) and 10 microgram per ml laminin (Cultrex,

Trevigen). All cultures were maintained in NB+ with 50 ng/ml NT-3 or NGF (Peprotech). To infect DRG

neurons with lentivirus, serum-free lentiviral stocks were diluted with NB+ and cells at the time of plating.

Virus containing medium was replaced 3 hours after plating. For starvation, cells were washed twice with

Neurobasal medium and were cultured for either 4 hours or for 16-20 hours in NB+ medium without

neurotrophin. In longer starvation experiments, 50 M BAF (Boc-Asp(OMe)-FMK, lMP Biomedicals) and

100 ng/ml anti-NT-3 (R&D Systems) was included in the media. For short-term NT-3 stimulation

experiments, cultures were pretreated with drugs for 30 minutes prior to stimulation with 50 ng/ml NT-3.

Explant cultures were prepared from E12.5 timed pregnant females by placing thoracic or lumbar DRG in

three-dimensional collagen gel (Becton-Dickinson) as described. When the determination of embryo

genotype was required, DRGs from an individual embryo were pooled in ice-cold Hibernate-E (BrainBits)

while PCR analysis was performed. Ganglia were isolated from 4 control and 4 mutant embryos from

three different litters, and explants were incubated in NB+ with NT-3 or NGF for 2 days followed by

fixation with 4% PFA and immunohistochemical staining with TUJ1. Axon outgrowth was measured by

taking the maximal diameter and subtracting the distance occupied by the ganglion. Unpaired t-test was

performed in GraphPad Prism (GraphPad software, La Jolla, CA)

Nucleofection of dissociated DRG cultures with SAD-A constructs (1 microgram SAD-A with 0.2

microgram pCAGs-myrYFP) was performed using the Amaxa mouse neuron nucleofector kit and the

Amaxa series 2 nucleofector (program O-003). After nucleofection, cells were recovered in Opti-MEM

media with 10% bovine growth serum and plated on glass coverslips coated with Poly-D lysine and

laminin in media containing 5 ng/ml NT-3. Cells were fixed after 2DIV and were immunostained for GFP

and TUJ1. Axonal tracing of neurons from three independent experiments was performed with the

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Simple Neurite Tracer plugin in ImageJ and total neurite length and branch number were quantified as

described (Lentz et al., 1999). One-way ANOVA with post hoc Tukey’s multiple comparisons test was

performed using GraphPad Prism.

Cell culture and lentivirus production

HeLa and 293T cells were cultured and transfected as described (Barnes et al., 2007). For NT-3

stimulation of TrkC-transfected HeLa cells, cells were trypsinized and re-seeded into 48 well culture

dishes 2 days post-transfection. After attachment, cells were serum starved for 4-6 hours and then

treated with NT-3 (50 ng/ml). For lentivirus production, 293T cells were grown in Opti-MEM + 2% FBS for

24 hours prior to transfection. Serum free lentiviral stocks were made by transfection of 293T cells with

lentiviral backbone plasmid, psPAX2 and pMDVSV-G using TransIT293 (Mirus) according to the

manufacturer’s specifications. Shortly after transfection, the media was changed to Opti-MEM without

serum. Virus containing supernatants were collected 48 hours later, filtered through a 0.45 micron filter

and snap frozen in aliquots.

RT-PCR

Total RNA was isolated from cultured DRG neurons using the RNAeasy kit (Qiagen). Equal amounts of

total RNA were used for reverse transcription using the Superscript III kit (Invitrogen); reverse

transcriptase enzyme was omitted in negative control samples. Triplicate qPCR reactions were

performed on a Stratagene MX3000P thermal cycler with Perfecta SYBR Green Fastmix, low Rox

(Quanta Biosciences). Primers used for amplification were: SAD-A (SAD-A F111: 5’-

CCTACCGGCTGGAGAAGAC-3’; SAD-A R237: 5’-AGGACCGACTCACTGAGCTT-3’); SAD-B (SAD-B

F77: 5’-AGGGAGAAGCTGTCGGAATC-3’; SAD-B R185: 5’-TGTTCTCGTAGACGTCGTGG-3’) 3

tubulin (TUBB3 F35: 5’-AGTCAGCATGAGGGAGATCG-3’; TUBB3 R159: 5’-

AGTCCCCTACATAGTTGCCG-3’). The Ct method was used to calculate expression levels in NT-3

starved neurons relative to samples treated with NT-3 with 3 tubulin used to normalize. Data are from

three independent experiments.

Biochemical Analysis

For direct analysis of protein lysates, cells were lysed in a denaturing buffer as described (Barnes et al.,

2007). For immunoprecipitation experiments, 1 x106 cells were lysed on ice in 20mM Tris-HCl, 150 mM

NaCl, 1% Triton X-100, 0.1% SDS, 1 mM DTT and protease and phosphatase inhibitors (Complete

protease and Phos-Stop phosphatase inhibitors, Roche). After centrifugation at 13,000 x g for 30 minutes

at 4°C, 15 microliters of 3F10 agarose beads (Roche) were added to equal amounts of lysate.

Immunoprecipitation reactions were rocked at 4°C for 3 hours and beads were washed 3 times with 20

mM Tris-HCl, 0.3 M NaCl, 0.1% Triton X-100 and were either resuspended in reducing sample buffer or

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washed once with kinase reaction buffer (KB: 20 mM Tris-HCl, 10 mM MgOAc, 1mM DTT, 0.1% Triton X-

100). Kinase assays were then performed by resuspending beads in 30 microliters of KB with 1 mM ATP

and 50 ng purified LKB1/STRAD/MO25 (Millipore). Reactions were incubated at room temperature with

shaking and aliquots of the reactions were taken at indicated times and mixed with 2X reducing sample

buffer. Expression and purification of GST-tagged SAD proteins in E. coli was performed as described

(Kim et al., 2008). Kinase assays with purified SAD proteins were performed as above with purified

LKB1/STRAD/MO25 or TAK1/TAB1 proteins (Millipore). Lambda protein phosphatase (NEB) was used

according to manufacturer’s instructions.

SDS-PAGE (Acrylamide concentrations in resolving gels of either 8% or 4-15% gradient) and

immunoblotting was performed as described (Barnes et al., 2007) and made use of the following

antibodies: SAD-A (mouse mAb 8H11, 1:10), anti-SAD-B (1:8000) (Kishi et al., 2005), anti-pSAD (ALT)

1:5000 (Barnes et al., 2007), anti-GFP (Abcam and Epitomics, 1:2000), rat mAb anti-HA (Roche 3F10,

1:2000), mouse mAb TUJ1 (Covance, 1:10000), anti-LKB1 (Millipore, 1:2000), anti-pTau S262 (Abcam,

1:2000). The following antibodies were purchased from Cell Signaling Technologies: anti-pERK1/2

(1:4000), anti-pAKT S473 (1:4000), anti-pS6 S235/236 (1:4000), anti-ERK1/2 (1:2000), anti-S6 (1:2000),

anti-TAK1 (1:1000), anti-TAB1 (1:1000), mouse anti-alpha tubulin (1:5000), mouse mAb pTP-101

(1:3000). Horseradish peroxidase conjugated secondary antibodies were from Jackson Immunoresearch.

Chemiluminescence detection was performed with SuperSignal West Pico chemiluminescence substrate

(Thermo) and signals were detected with Kodak BioMax Light film or with a Bio-Rad ChemiDoc XRS

system with QuantityOne software (Bio-Rad). Dual color immunoblotting was performed with Immobilon-

FL PVDF membrane (Millipore) and fluorescently conjugated secondary antibodies. Membranes were

scanned on a STORM imaging system (GE Healthcare). All biochemical experiments were performed a

minimum of three times and representative images of immunoblots are shown. Quantification of

GFP::SAD-A abundance was performed using densitometry in ImageJ. GFP signals were normalized to

non-saturating signals of TUJ1 from the same sample and relative densities of GFP signals from NT-3

deprived samples are expressed as a percentage of those from NT-3 treated samples.

Chemicals used in this study were obtained from the following manufacturer’s: EMD Biosciences:

PD325901 (used for long-term inhibition of MEK1/2 due to instability of U0126), Roscovitine; Cell

Signaling Technology: U0126, LY294002, Rapamycin,; Tocris: U73122; Sigma: A23187.

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Rossi, J., Balthasar, N., Olson, D., Scott, M., Berglund, E., Lee, C.E., Choi, M.J., Lauzon, D., Lowell, B.B., and Elmquist, J.K. (2011). Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis. Cell Metabolism 13, 195-204.