Phospho mtor s2448

Cells were treated with kaempferol, washed with ice-cold PBS and then lysed with RIPA buffer supplemented with phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor. Cell lysates were centrifuged at 15,000 rpm and 4 °C for 15 min, and protein concentrations were determined with a BCA protein assay kit (Beyotime, Shanghai, China). Subsequently, equal amounts of protein samples were separated by electrophoresis using 6, 10 or 12.5% SDS-PAGE Gel Fast Preparation Kits (Epizyme, Shanghai, China) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with antibodies against the following proteins: β-actin (1:1000; Proteintech), Akt (1:1000; Proteintech), phospho-Akt (Ser473) (1:1000; CST), TGM2 (1:1000; CST), Kelch-like ECH-associated protein 1 (KEAP1) (1:1000; Proteintech), NF-E2-related factor 2 (NRF2) (1:1000; Proteintech), mTOR (1:1000; Proteintech), phospho-mTOR (S2448) (1:1000; Abcam), Bax (1:1000; Proteintech), Bcl2 (1:1000; Proteintech), Caspase 3 (1:500; Proteintech), PARP1 (1:1000; Proteintech), and cleaved PARP1 (1:1000; Abcam).

Primary antibodies used for Western blot detection of proteins were obtained from Cell Signaling Technology, unless specified otherwise: phospho-AKT S473 (#9271), phospho-JAK1 (#3331), phospho-STAT5 (#9351), phospho-MEK1/2 (#9154), phospho-ERK1/2 (#4370), phospho-mTOR S2448 (#2971), phospho-p70S6 Kinase T421/S424 (#9204), BAD (Abcam #AB32455), phospho-BAD S136 (#4366), BIM (Abcam #AB15184), phospho-BIM S55 (#4550), phospho-BIM S69 (#4581), phospho-CREB S133 (#9198), phospho-ATF1 (#9198), BCL2 (Santa Cruz Biotechnology #sc-130308), BCLXL (#2764), MCL1 (Sigma #HPA008455), PUMA (Abcam #AB33906), cMYB (#12319), phospho-IKKab S176/S177 (#2978), phospho-IKKab S176/S180 (#2697), GSK3AB (#5676), phospho-GSK3AB S21/9 (#9331), phospho-p38/MAPK T180/Y182 (#4511), GCR (Santa Cruz Biotechnology #sc-1003), phospho-GCR S211 (Abcam #AB3579), DYKDDDDK Tag Antibody (#2368), CD127 (IL7R) (R&D Systems #MAB306), RAS (Millipore #05–516), and β-actin (Abcam #AB6276). Following staining of Western blots using IRDye fluorescent secondary antibodies, fluorescent intensities were scanned and quantified using an Odyssey imager (LI-COR) and normalized to β-actin expression levels.

Cells were harvested and lysed using lysis buffer for 15 min at 4°C. The protein concentration was assessed using a bicinchoninic acid (BCA) assay. Western blotting was carried out as described previously (21 (link)). The primary antibodies were as follows: phospho-AKT S437 (4060T; Cell Signaling Technology), total AKT (4691T; Cell Signaling Technology), phospho-mTOR S2448 (ab109268; Abcam), total mTOR (ab32028; Abcam), phospho-ERK1/2 T202/Y204 (#4370; Cell Signaling Technology), total ERK1/2 (#4695; Cell Signaling Technology), and β-actin (ab8226; Abcam).