Anti tsc2

Antibodies used for immunoblotting included anti-sestrin2 obtained from Proteintech Group, anti-phospho-p70 S6 Kinase, anti-phospho-S6 Ribosomal Protein, anti-S6 Ribosomal Protein, anti-E-cadherin, anti-N-cadherin, and anti-TSC2 from Cell Signaling Technology, anti-p70 S6 Kinase from Santa Cruz Biotechnology, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Aviva Systems Biology, and anti-β-Actin from Developmental Studies Hybridoma Bank. Rapamycin was obtained from LC Laboratories. Torin 1 was purchased from Cayman Chemical. N-acetyl cysteine (NAC) was purchased from Sigma-Aldrich.

Proteins were extracted from RMCs using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, and protease cocktail at 1 μg/mL). Protein concentrations were determined using a BCA kit (Thermo Scientific, Rockford, AL, USA). Equal amounts of protein (60 µg) were separated by SDS-PAGE on a 6%, 10%, or 12% acrylamide gel and then transferred onto a nitrocellulose membrane. After the membrane had been blocked with non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature, it was probed with the following primary antibodies overnight at 4°C: anti-VDR (catalogue no.: ab109234, Abcam, USA), anti-DDIT4 (catalogue no.: NBP1–77321, Novus Biologicals, USA), anti-TSC1 (catalogue no.: 6935S, Cell Signaling Technology, USA), anti-TSC2 (catalogue no.: 4308P, Cell Signaling Technology), anti-Rheb (catalogue no.: 13879S, Cell Signaling Technology), anti-mTOR (catalogue no.: Ab51044, Abcam), anti-4E-BP1 (catalogue no.: ab2606, Abcam), and anti-p70S6K (catalogue no.: ab32359, Abcam). After extensive washing, the membranes were incubated with Dylight anti-rabbit IgG secondary antibody. The antigens were visualized using the Odyssey infrared imaging system (LI-COR Biotechnology, Nebraska, USA). The results were expressed as the relative intensity (RI) intensity (adjusted to that of β-actin) of each band.

The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): anti-pan-acetylated lysine (#9441), anti-MAP1LC3/LC3 (#4108), anti-RPS6KB/p70S6K (#9202), anti-phospho-RPS6KB/p70S6K (Thr389) (#9205), anti-TSC2 (#9442), anti-phospho-ULK1 (Ser 757) (#14202), anti-ULK1 (#8054), anti-phospho-DNM1L/Drp1 (Ser 616) (#3455), anti-phospho-MAPK3/ERK1-MAPK1/ERK2 (Thr 202/Tyr 204) (#9101), anti-MAPK3/ERK1-MAPK1/ERK2 (#9102), and anti-P-EIF2A/P-eIF2-α (Ser 51) (#9721). From Mitosciences: anti-RIESKE (MS305/C0183). From Abcam: anti-MFN2 (ab56889) and anti-HADHA (ab54477). From Sigma-Aldrich: anti-ACTB/β-actin (A5316). From Santa Cruz Biotechnology: anti-GFP (sc-9996), anti-TSC2 (sc-893), anti-TOMM20 (sc-17764), and anti-MFN1 (sc-50330). From Merck-Millipore: Nitrotyrosine 06-284. Other antibodies were used as follows: anti-mono- and polyubiquitinylated proteins FK2 conjugated with peroxidase (HRP) or FK2H from Enzo Life Sciences (BML-PW0150), anti-OPA1 (612606), and anti-DNM1L/Drp1 (611112) from BD Biosciences. Chloroquine C6628, propidium iodide (P4170), and thapsigargin (T9033) were from Sigma-Aldrich; rapamycin (553210) and resveratrol (R5010) were from Merck; and geneticin (G418) was from Santa Cruz (sc-29065).

Protein lysates from cells and tissue samples were prepared using the CelLytic M Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO). The proteins were resolved on an SDS-PAGE and then transferred to a PVDF membrane (Pall Corp., Port Washington, NY). The signal was visualized using an appropriate antibody and the Immobilon Western Chemiluminescent HRP substrate (Milipore, Billerica, MA). The anti-β-actin antibody (Cat# A5441) purchased from Sigma-Aldrich (St. Louis, MO) was used as a loading control. Antibodies such as anti-TSC1 (Cat# 6935S), anti-TSC2 (Cat# 3612), anti-phospho-p70S6 Kinase (Thr389) (Cat# 9205), anti-p70S6 Kinase (49D7) (Cat# 2708), anti-phospho-ULK1 (Ser757) (Cat# 6888) and anti-SQSTM1/p62 (Cat# 5114) were purchased from Cell Signalling Technologies (Danvers, MA). The anti-mouse HRP-conjugated secondary antibody (Cat# HP06) and anti-rabbit HRP-conjugated secondary antibody (Cat# HP03) were purchased from Bangalore Genei (Bangalore, India). The anti-phospho-TSC2 (S939) (Cat# ab52962) was purchased from Abcam (Cambridge, MA).

Cells were lysed in SDS buffer. The protein concentration was measured by BCA assay kit (Thermo). Equal amounts of cell lysates were loaded, blotted onto a PVDF membrane, and probed with the following primary antibodies: anti-PTEN(Cell signaling), anti-Akt(abcam), anti-p-Akt(Cell signaling), anti-PI3K(Abcam), anti-TSC1(Cell signaling), anti-TSC2(Cell signaling), anti-S6K(Cell signaling), ant-mTOR(Abcam), anti-FOXO1(Abcam), anti-GSK3β(Cell signaling), anti-p-GSK3β(Cell signaling), anti-β-Catenin(Cell signaling), anti-p-β-Catenin(Santa Cruz), anti-GAPDH (Cell signaling), and anti-β-Actin (Santa Cruz) was used as loading controls. After incubation with the appropriate secondary antibodies, signals were visualized by enhanced chemiluminescence (GE systems).