Anti pakt thr308

Fresh tissues and cells were lysed with cell lysis buffer (Beyotime Biotechnology) and western blot was performed as described previously [18 (link)]. Briefly, 40 μg total proteins were applied to separation with SDS–PAGE gel. After the electrophoresis, the proteins were transferred to PVDF membranes (Millipore), followed by blocking in the TBST buffer containing 5% fat-free milk. The membranes were then incubated with indicated antibodies overnight at 4 °C, and then washed and incubated with HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 h at room temperature and finally visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following antibodies were used in this work: Anti-GAPDH (Cell Signaling Technology), anti-JMJD2A (Cell Signaling Technology), anti-Histone H3 (Santa Cruz Biotechnology), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-mTOR (Cell Signaling Technology), anti-p-mTOR (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt Thr308 (Cell Signaling Technology), anti-S6K1 (Cell Signaling Technology), anti-p-S6K1 (Cell Signaling Technology).

Retina tissue samples were collected and prepared as described previously [31 (link)]. Briefly, intact retinas were homogenized in a Tris lysis buffer including (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, and 1 Na₃VO₄; pH 7.5. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The primary antibodies used in this study were anti-glucose transporter 4 (anti-Glut4; Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated protein kinase B (AKT) at thr 308 (anti-pAKT-thr308; Cell Signaling Technology), anti-VGCCα1D (Alomone, Jerusalem, Israel), and anti-ERK (total ERK, used for loading control; Santa Cruz Biochemicals, Santa Cruz, CA, USA). Blots were visualized using appropriate secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology) and an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). Relative protein expressions for all proteins involved in this study are reported as a ratio to total ERK. Band intensities were quantified by densitometry using Scion Image (NIH, Bethesda, MD, USA). All measurements were repeated at least 3 times.

All chemicals were obtained from Sigma-Aldrich unless otherwise specified. The following antibodies were used in this study: rabbit anti-UCP1 (Abcam, ab155117, GR3233606-10 1:2000), anti-Parvalbumin (ABclonal, A13538, Lot0054370201 1:1000), anti-Transferrin (Abbkine, ABM40235, 1:1000), anti-pSTST6 (Cell Signaling Tech, 56554, 1:2000), anti-STAT6 (Cell Signaling Tech, 9362, 1:2000), anti-pAKT (Ser473) (Cell Signaling Tech, 4060, 1:2000), anti-pAKT (Thr308) (Cell Signaling Tech, 13038,1:2000), anti-AKT (Cell Signaling Tech, 2920, 1:2000), anti-GAPDH (Santa Cruz, sc32233, 1:1000), anti-GFP (Proteintech, 50430-2-AP, 1:1000), anti-pERK (Cell Signaling Tech, 4370, 1:2000), anti-ERK (Cell Signaling Tech, 4695, 1:2000), anti-pPKC (Abcam, ab180848, 1:2000), anti-PKC (Abcam, ab179522, 1:2000), anti-p4EBP1 (Cell Signaling Tech, 2855, 1:2000), anti-4EBP1 (Cell Signaling Tech, 9452, 1:2000), anti-ACTB (Sigma Aldrich, A3854, 1:10000), anti-Flag (Sigma-Aldrich, F7425, 1:10000), HRP-conjugated goat anti-Rabbit IgG (Cell Signaling Tech, 7074, 1:10000), anti-pGSK-3β (Cell Signaling Tech, 5558, 1:2000), anti-GSK-3β (Cell Signaling Tech, 12456, 1:2000). Rictor antibody (Abcam, ab70374, 1:100), mTOR antibody (Cell Signaling Tech, 2983, 1:100) (Supplementary Table 2).

Western-blot assays were performed as previously described [11 (link)]. Following antibodies were used. Anti-p-AKT (Ser473) (#4051, Cell signaling), Anti-p-AKT (Thr308) (#4056, Cell signaling), Anti-AKT (Sc-1618, Santa Cruz). Anti-Flag-M2 (F1804, Sigma), Anti-β-Actin (A5441, Sigma). GLS2 antibody was prepared as previously described [11 (link)]. The protein levels were quantified by digitalization of the X-ray film and analyzed with Scion Image software (Scion Corporation, Frederick, MD).

Antibodies used in this study include Anti-SGK196 (ab57908, Abcam, Cambridge, UK); Anti-GSK3β (A0480, Abclonal, Wuhan, China), Anti-p-GSK3β-S9 (AP0039, Abclonal); Anti-RPN1 (sc48367, Santa Cruz, Dallas, TX); Anti-p-AKT-Ser473 (#4058, Cell Signaling Technology, Danvers, MA), Anti-p-AKT-Thr308 (#13038, Cell Signaling Technology), Anti-AKT(pan) (#2920, Cell Signaling Technology), Anti-Snail (#3879, Cell Signaling Technology); Anti-HA (H6908, Sigma-ALDRICH, MO), Anti-Flag (F1804, Sigma-ALDRICH), Vimentin (sc-66001,Santa Cruz), α-dystroglycan (VIA4) (sc-53986, Santa Cruz) and Anti-GAPDH (HC301-01, TRANSGEN BIOTECH, Beijing, Shanghai). All antibodies for Western blotting were used at dilution 1:1000. For Immunofluorescence assays, Anti-HA (H6908, Sigma-ALDRICH) was used at dilution 1:100. Other reagents include DMEM medium (11965-084, Gibco), RPMI 1640 medium (11875-085, Gibco), Cell Counting Kit reagents (40203ES60, YEASEN), puromycin (P8230, Solarbio, Beijing, China), LY294002 (L9908, Sigma-ALDRICH), CHX (5087390001, Sigma-ALDRICH), Protease inhibitor Cocktail (EDTA-Free, 100x in DMSO, 20124ES03, YEASEN), Endo H (# P0702S, New England Biolabs, Ipswich, MA), and PNGase F (# P0705S, New England Biolabs).

Protein from cell cultures were extracted by freeze-and-thaw cycles. Muscles were cut into 40 slices, 20 μm thick and lysed with 100 μl of lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 10 mM MgCl2, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 2% SDS, 1% Triton from Sigma-Aldrich®, 1:50 Complete Phosphatase inhibitor from Roche®). The total protein concentration was determined using BCA kit (Abcam®, Prodotti Gianni, Milano, Italy). Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and then western blotting. Primary antibodies were anti-p-AKT (Thr308, cat# 13038), anti-p-mTOR (Ser2448, cat# 2971), anti-p-S6 (Ser 235/236, cat# 4858), monoclonal anti-GAPDH (cat# 5174), anti-LC3 (cat# 4108), anti-P62 (cat# 8025, all from Cell Signaling®; dilution 1:1000), anti-Lys63 (cat# 05-1308), and anti-Lys48 (cat# 05-1307, both from Millipore®; dilution 1:500) (25 (link)). Immunoreactive bands were detected using proper HRP-secondary reagent and Clarity Western ECL Substrate (Bio-Rad).

Whole-cell lysates were prepared by using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Roche, Nutley, USA), and proteins were quantified by using a bicinchoninic acid (BCA) protein quantitation kit (Thermo Scientific). Ten micrograms of whole-cell lysate per lane was used for immunoblotting. The following antibodies were used: anti-KRAS (Sigma-Aldrich, St. Louis, USA), anti-DCBLD2 (Cell Signaling, Danvers, USA), anti-p-AKT-Ser473 (Cell Signaling), anti-p-AKT-Thr308 (Cell Signaling), anti-total-AKT (Cell Signaling), anti-PIK3R2 (Santa Cruz, Dallas, USA), anti-PLK2 (Santa Cruz), and anti-UBQLN2 (Millipore, Billerica, USA) at 1:1000 dilution; anti-β-Actin and anti-GAPDH (Cell Signaling) at a dilution of 1:2000.