Anti p p70s6k

Proteins were extracted from more than 10⁶ harvested BMSCs according to the kit protocol (KeyGen Biotech, China). [46 (link)] BMSCs were digested in a lysis buffer cocktail and centrifuged at 12000 g for 15 minutes at 4 °C to get the supernatant as the total protein. 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used for electrophoresis of protein (20 μg) with loading buffer and then blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline tween 20, TBST, and then incubated overnight at 4°C with primary antibodies; anti-IHH, anti-P53, anti-P16, anti-PI3K, anti-p-PI3K, anti-Akt 1, anti-p-Akt 1, anti-NF-κB, anti-p-NF-κB-p65, anti-STAT3, anti-p-STAT3, anti-4EBP1, anti-p- 4EBP1, anti-p70S6K, anti-p-p70S6K and anti-β-actin (1:1000), all from Abcam, USA. The membranes were incubated for 1hour at RT with secondary antibody conjugated with horseradish peroxidase. Finally, TBST-washed membranes were treated with enhanced chemiluminescent (ECL) for detection (Bio-Rad, USA). Image Lab detection system (Bio-Rad, USA) was used for protein bands imaging and analysis.

TEOA was obtained from college of pharmacy, Zhejiang University (Zhejiang, China) and dissolved in DMSO. N-acetylcysteine (NAC), Chloroquine (CQ), 3-Methyladenine (3MA) and Rapamycin were purchased from Medchem Express (MCE, United States). DAPI was purchased from Beyotime (Shanghai, China). The following primary antibodies were used: anti-GAPDH (Abcam ab181602) (1:2,000), anti-Catalase (Abcam ab76024) (1:1,000), anti-MnSOD (Abcam ab68155) (1:2,000), anti-LC3 (Sigma L7543) (1:1,000), anti-p-mTOR (Abcam ab109268) (1:2,000), anti-mTOR (Abcam ab134903) (1:1,000), anti-p-p70S6K (Abcam ab131436) (1:1,000), anti-p70S6K (Abcam ab32529) (1:1,000), anti-p-S6 (CST 4858S) (1:2,000), anti-S6 (CST 2217s) (1:1,000), anti-p-4EBP1 (CST 9459S) (1:1,000), anti-4EBP 1 (CST 9644S) (1:1,000), anti-ATG5 (Abcam ab68155) (1:2,000). The related HRP-conjugated secondary antibody was purchased from Beyotime (Shanghai, China).