Smithson and Gutmann, Supplemental Materials

Supplemental Materials and Methods

Supplemental Figures and Figure Legends

Supplemental Fig. S1, related to Figure 1

Supplemental Fig. S2, related to Figure 2

Supplemental Fig. S3, related to Figure 3

Supplemental Fig. S4, related to Figure 4

Supplemental Tables

Supplemental Table S1: Antibodies

Supplemental Table S2: Constructs

Supplemental Table S3: Proteomic analysis of WT astrocyte mTOR-immunoprecipitates (excel

file)

Supplemental Experimental Procedures

Cell cultures/lines. Wild-type (WT) neural stem cells were generated from the brainstems of

PN1-2 mouse pups and maintained in low glucose neurobasal media supplemented with FGF

(20ng/ml), EGF (20ng/ml), 1% N2, 2% B27, 2mM L-glutamine, 0.05mM β-mercaptoethanol,

and 0.05% penicillin/streptomycin. Neural stem cells were passaged thrice and serum starved for

24 hours prior to analysis.

The mouse K4622 glioma cell line was derived from a C57Bl/6 Nf1+/-; p53+/- (NPCis) mouse

that formed an Nf1- and p53-deficient high-grade astrocytoma, as previously described (Banerjee

et al., 2011). These cells were maintained in DMEM supplemented with 10% heat-inactivated

fetal bovine serum (HI-FBS) and 1% penicillin/streptomycin (Gibco).

Tissues. Neural and non-neural tissues were harvested from 1-month-old wild-type C57BL/6

mice (n=3) and snapped frozen in liquid nitrogen prior to western blot analysis.

Rac1 activity assay. Active Rac1 was assayed using the Active Rac1 Detection Kit (Cell

Signaling, #8815). Nf1-/- astrocytes infected with shGIT1 were lysed in 400µl of 1x

Lysis/Binding/Wash Buffer (from Kit) with protease inhibitors. For positive and negative

controls, 10mM GTPγS (positive) and 100mM GDP (negative) plus 0.5M EDTA were used,

respectively. Active Rac1 was detected following a one-hour incubation period at 4°C with GST-

PAK1-PBD. Lysates were washed thrice with 1X Cell Lysis/Binding/Wash buffer and eluted in

2X Laemmli buffer. Samples were resolved by 12% SDS-PAGE, transferred to PVDF and

analyzed by western blotting with a supplied Rac1 mouse monoclonal antibody (1:1000).

Horseradish peroxidase-conjugated affinity purified horse anti-mouse (#7076) secondary

antibodies (1:5000, Cell Signaling Technology) were used for enhanced chemiluminescence-

based detection (Pierce).

Supplemental Figure S1. Protor-1 and Deptor are not expressed in primary mouse

astrocytes and fibroblasts. (A) A representative immunoblot (IB) reveals an absence of Protor-

1 and Deptor in mouse astrocytes and fibroblasts. In contrast, mouse kidney and liver express

both of these proteins.

Supplemental Figure S2. GIT1 is enriched in neural tissues. (A) A representative immunoblot

(IB) demonstrates the GIT1-binding protein, Rho guanine nucleotide exchange factor 7 (β-PIX),

in mTOR immunoprecipitates (IPs). Normal rabbit IgG (Rb IgG) was used as a control for non-

specific binding. (B) GIT1 binding to mTOR is also observed in postnatal day 1 (PD1) and 1-

month-old mouse brainstem tissue. (C) GIT1 binding to mTOR is observed in neural stem cells

(NSCs, 200mg input), as well as in HEK293T cells (293T, 800mg input; in the presence (+) or

absence (-) of fetal bovine serum [FBS]). GIT1 binding to mTOR in 293T cells was estimated at

100-fold lower than observed in NSCs. (D) GIT1 protein expression predominates in neural

tissues (cortex, cerebellum, olfactory bulb). (E) A representative IB of GIT1 IPs reveals no

mSIN1 (mTORC2) and PRAS40 (mTORC1) binding.

Supplemental Figure S3. GIT1 binding to mTOR is not regulated by phosphorylation of

GIT1 or mTOR in astrocytes. (A, B) Representative IBs demonstrate no change in the

phosphorylation of GIT1 (Tyr554) or mTOR (Ser2448 or Ser2481) in Nf1-deficient (Nf1-/-) brainstem

astrocytes relative to WT controls. α-tubulin was included as an internal protein loading control.

(C) AKT inhibitor (MK2206) treatment of WT astrocytes does not affect GIT1 binding to

mTOR. AKT and mTOR inhibition were confirmed by reduced PRAS40 (phospho-

PRAS40Thr246) and S6 (phospho-S6Ser240-244) activation, respectively.

Supplemental Figure S4. GIT1 is required for mTORC1- and mTORC2-independent

astrocyte growth (A) shRNAi (TRC0000346504) GIT1 knockdown (shGit1KD) restores Nf1-/-

astrocyte proliferation to levels observed in WT astrocytes. (B) shGit1 KD results in increased

caspase-3 cleavage in Nf1-/- astrocytes. No change in LC3 expression was observed. α-tubulin

was included as an internal protein loading control. (C) Following shGit1 KD in WT astrocytes,

no change in caspase-3 cleavage or the percentage of TUNEL+ cells was observed. (D) shGit1

KD does not change Rac1 activity in Nf1-/- astrocytes. (E) mTORC1 (4EBP1) or C2 (PKCα/β,

SGK1) effector activation is similar in WT and Nf1-deficient astrocytes. shGit1 KD in Nf1-/-

astrocytes does not change mTORC1 (4EBP1) or mTORC2 (PKCα/β-II, SGK1) effector

activation (phospho-4EBP1Ser65, phospho-PKCα/β-IIThr638/641, and phospho-SGK1Ser78). (**)

p<0.005.

Supplemental Table S1. Antibodies.

Antibody Company Catalog # Dilution Species Application

AKT Cell Signaling 9272 1:1000 rabbit WBPhospho-AKTThr308 Cell Signaling 9275 1:500 rabbit WB

β-PIX Cell Signaling 4515 1:500 rabbit WBCleaved Caspase 3 Cell Signaling 9664 1:500 rabbit WB

Deptor Abcam ab941841 1:500 rabbit WB4EBP1 Cell Signaling 9452 1:1000 rabbit WB

Phospho-4EBP1Ser65 Cell Signaling 9451 1:1000 rabbit WBERK Cell Signaling 9102 1:1000 rabbit WB

Phospho-ERK1/2Thr202/

Tyr204 Cell Signaling 4370 1:8000 rabbit WB

FLAG epitope Cell Signaling 2368 1:1000, 1:100 rabbit WB, IPGAPDH Abcam ab8245 1:10,000 mouse WB

GβL/mLST8 Cell Signaling 3274 1:1000 rabbit WBGIT1 Cell Signaling 2919 1:1000, 1:50 rabbit WB, IP

Phospho-GIT1Tyr554 Abgent AP3553a 1:500 rabbit WBGSK-3β Cell Signaling 12456 1:500 rabbit WB

Phospho-GSK-3βSer9 Cell Signaling 9336 1:500 rabbit WBLC3A/B Cell Signaling 4108 1:500 rabbit WBmTOR Cell Signaling 2972 1:1000, 1:100 rabbit WB, IP

Phospho-mTORSer2448 Cell Signaling 5536 1:2000 rabbit WBPhospho-mTORSer2481 Cell Signaling 2974 1:1000 rabbit WB

myc epitope Cell Signaling 2278 1:1000 rabbit WB, IPNeurofibromin Santa Cruz sc-67 1:200 mouse WB

NME1 Cell Signaling 3345 1:500 rabbit WBNormal rabbit IgG Cell Signaling 2729 1:100 rabbit IP

PAK3 Cell Signaling 2609 1:500 rabbit WBPKCα Cell Signaling 2056 1:1000 rabbit WB

Phospho-PKCα/β-IIThr638/641 Cell Signaling 9375 1:1000 rabbit WB

PRAS40 Cell Signaling 2691 1:1000 rabbit WBPhospho-PRAS40Thr246 Cell Signaling 2997 1:1000 rabbit WB

Protor-1 Abcam ab154645 1:500 rabbit WBRac1 Cell Signaling 8631 1:1000 mouse WB

Raptor Cell Signaling 2280 1:1000 rabbit WB

Raptor Bethyl Laboratories

A300-553A 1:50 rabbit IP

RhoC Cell Signaling 3430 1:1000 rabbit WBRictor Cell Signaling 9476 1:1000, 1:50 rabbit WB, IPmSIN1 Abcam ab71152 1:500 rabbit WB

Phospho-S6Ser240/244 Cell Signaling 2215 1:10,000 rabbit WBS6 Cell Signaling 2217 1:10,000 rabbit WB

SGK1 Cell Signaling 12103 1:500 rabbit WBPhospho-S6K1Ser78 Cell Signaling 5599 1:500 rabbit WB

TOP2α Cell Signaling 4733 1:500 rabbit WBTGM2 Cell Signaling 3557 1:500 rabbit WB

α-tubulin Sigma-Aldrich T9026 1:10,000 mouse WB

YAP Cell Signaling 4912 1:500 rabbit WBPhospho-YAPSer127 Cell Signaling 13008 1:500 rabbit WB

Supplemental Table S2: Constructs

Construct Company/Provided by Product # Ref.

myc-mTOR full length Addgene;David Sabatini 1861 Sarbassov et al., 2004

myc-mTOR 1-1482 Addgene;David Sabatini 21745 Kim et al., 2003

myc-mTOR 1271-2008 Addgene;David Sabatini 21746 Kim et al., 2003

myc-mTOR 1750-2549 Addgene;David Sabatini 21747 Kim et al., 2003

FLAG-GIT1 full length Mark Sowden n/a Yin et al., 2004FLAG-GIT1 1-420 Mark Sowden n/a Yin et al., 2004

FLAG-GIT1 250-770 Mark Sowden n/a Yin et al., 2004FLAG-GIT1 420-770 Mark Sowden n/a Yin et al., 2004FLAG-GIT1 delSHD Mark Sowden n/a Yin et al., 2004

FLAG-pCMVg Mark Sowden n/a Yin et al., 2004

shGit1-1 Sigma-Aldrich TRC0000346504 n/a

shGit1-2 Sigma-Aldrich TRCN0000346581 n/a

shGit1-3 Sigma-Aldrich TRCN0000106120 n/a

Supplemental Table S3. Proteomic analysis of mTOR-immunoprecipitation in WT astrocytes

(Excel File).