Anti p mtor antibody

For western blotting, total protein was extracted from cardiac tissue and myocyte cells using a RIPA buffer and separated by SDS-PAGE, and the protein concentration was examined with a BCA protein assay kit (KGP902; Keygen, China). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk powder for 1 h. The membranes were probed with the following primary antibodies: anti-IKKε antibody (1 : 1000; Cell Signaling, #2690); anti-LC3 antibody (1 : 2000; Abcam, USA, #ab51520); anti-ATG-5 antibody (1 : 1000; Cell Signaling, USA, #2630); anti-p-AKT antibody (1 : 1000; Cell Signaling, USA, #S473); anti-AKT antibody (1 : 1000; Cell Signaling, USA, #C67E7); anti-mTOR antibody (1 : 1000, Cell Signaling, USA, #7C10); and anti-p-mTOR antibody (1 : 1000; Cell Signaling, USA, #S2448). The following day, the PVDF membranes were incubated with secondary antibodies (1 : 5000; Cell Signaling) at room temperature for 1 h. Specific proteins were detected using an ECL reagent (GE Healthcare, Piscataway, NJ, USA) and captured on Hyperfilm (Amersham, GE Healthcare). The results were then analyzed using the ImageJ software for semiquantitation of the mean gray value of each blot.

Cultured cells or heart tissues were used to extract total protein with RIPA lysis buffer (Millipore) with protease inhibitor cocktail (Biomake). Next, standard Western blot was performed to determine the levels of interested proteins according to a protocol modified from previous studies [31 (link), 32 (link)]. The primary antibodies used for Western blot assay are :
Anti-IDO1 antibody (Abcam; 1:1000), anti-GAPDH antibody (Proteintech; 1:2000), anti-pAKT antibody (Cell Signaling Technology; 1:1000), anti-AKT antibody (Cell Signaling Technology; 1:1000), anti-pmTOR antibody (Cell Signaling Technology; 1:1000), anti-mTOR antibody (Cell Signaling Technology; 1:1000), anti-pS6K1 antibody (Cell Signaling Technology; 1:1000), anti-S6K1 antibody (Cell Signaling Technology; 1:1000). The secondary antibodies (1:5000) and ECL kit were purchased from Servicebio.

Total proteins were extracted from brain tissues and neurons with RIPA buffer (Beyotime, P0013) supplied with proteinase inhibitor cocktail (Roche, 04693124001) and phosphatase inhibitor cocktail (Sigma, P5726). Total protein (30 μg) was subjected to SDS-PAGE for protein separation. The proteins were transferred to PVDF membranes and blocked with 5% fat-free milk in TBST buffer, and then the membranes were incubated with individual primary antibodies overnight. Then the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshanjinqiao) for 2 h. Finally, the secondary antibodies were detected by SuperSignal™ Chemiluminescent HRP Substrates (Thermo Fisher, 32106). The following primary antibodies were used in the present study: anti-GAPDH antibody (Santa Cruz, sc-47724), anti-BCAT1 antibody (Novus Biological, NBP2-01826), anti-Tau antibody (Abcam, ab64193), anti-p-Tau antibody (Abcam, ab109390), anti-mTOR antibody (Cell Signaling Technology, 2983), anti-p-mTOR antibody (Cell Signaling Technology, 5536), anti-S6K1 antibody (Cell Signaling Technology, 9202), and anti-p-S6K1 antibody (Cell Signaling Technology, 9206).

Cells were lysed in RIPA buffer, and then lysates were centrifuged at 13,400
g for 10 min and quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Samples were separated on 8%‒12% SDS-PAGE gels and transferred to PVDF membranes (IPVH00010; Millipore, Billerica, USA). The membrane was blocked for 1 h at room temperature with 5% milk solution in TBS-Tween [50 mM Tris (pH 8.0), containing 150 mM NaCl and 0.1% Tween 20] before incubation with primary antibodies, including anti-SREBP2 antibody (Abcam), anti-IL-1β antibody (Proteintech), anti-mTOR antibody, and anti-pmTOR antibody (Cell Signaling Technology, Danvers, USA) at 4°C overnight. Subsequently, HRP-conjugated secondary antibody anti-rabbit IgG was added, followed by incubation at room temperature for 1 h. Western blots were visualized using chemiluminescence reagents (Sigma, St Louis, USA).

Anti-CHOP antibody (CS2895), anti-p-eIF2α antibody (CS3398), anti-eIF2α antibody (CS2103), anti-p-JunB antibody (CS3177), anti-p-Grp78 antibody (CS3177), anti-p-4E-BP1 antibody (CS9455), anti-p-mTOR antibody (CS2971), anti- mTOR antibody (CS2972), anti-p-p70 kinase antibody (CS9234), anti-p-S6RP(Ser235/236), antibody (CS4856), anti-p-S6RP (Ser240/244), antibody (CS5364), anti-S6RP antibody (CS2317), anti-Grp94 antibody (CS2104), anti-JunD antibody (CS5000), and anti-β-Actin antibody (CS3700) were purchased from Cell Signaling Technology (Boston, MA, USA); anti-XBP1 antibody (AB) was purchased from Abcam (Cambridge, MA, USA).
Chloroquine disulfate (S6628), thapsigargin (T9030), Fli06 (SML0975), tunicamycin (T7765), 5-aminoorotic acid (191213), teriflunomide (SML0936), and DFMO (D193) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and diluted in DMSO or in water according to the manufacturer recommendation.