P mtor

Total protein was extracted using RIPA buffer containing proteinase inhibitor (Best Biological, Jiangsu, China). Western blot was conducted as previously described [12 (link)]. The primary antibodies for P-ERK1, ERK1, NF-κB1, RELA, P-mTOR, mTOR, VEGFA, PD-L1, P-P13K, P13K, P-AKT, AKT, TNF-α, P-EGFR, EGFR and HIF-1α were purchased from Bioss (Beijing, China). Total protein level was normalized to β-actin.

The BMECs of each group were collected and the total protein was extracted by a total protein extraction kit (Beyotime). A BCA protein assay kit (Beyotime) was used to measure the protein concentration. A 60µg sample of each group was loaded onto 10% SDS-PAGE electrophoresis, then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with TBST containing 5% skim milk, followed by incubation with the primary antibodies of LC3 (Abcam, Cambridge, UK, dilution 1:1000), PI3K (Proteintech Group, Wuhan, China, dilution 1:2000), P62 (Abcam, Cambridge, UK, dilution 1:1000), p-Akt (Abcam, Cambridge, UK, dilution 1:5000), Akt (Proteintech Group, Wuhan, China, dilution 1:2000), p-mTOR (Bioss, Beijing, China, dilution 1:1000), mTOR (Bioss, Beijing, China, dilution 1:1000), and GADPH (Hangzhou Goodhere Biotechnology, China, dilution 1:1000) at 4 °C overnight, then washed with TBST for 10 min and incubated with secondary antibodies for 2 h at room temperature.
Finally, the result was visualized by a chemiluminescence detection system (FluorChem M, ProteinSimple, San Jose, CA, USA).

Total protein samples were obtained after lysing in RIPA lysis buffer (Yeasen, Shanghai, China). The concentration was examined by a BCA kit (Beyotime Biotechnology, Shanghai, China). The quantified protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Boston, MA, USA). After blocking in 5% skimmed milk, the membranes were probed with primary antibodies LC3II/I (bs-8878R, Bioss, Woburn, MA, USA), Beclin1 (bs-1353R, Bioss), ATG5 (bs-4005R, Bioss), ULK1 (bs-3602R, Bioss), p-mTOR (bs-3495R, Bioss), mTOR (bs-1992R, Bioss), p-P70S6K (#97,596, CST, MA, USA), P70S6K (#34,475, CST), NLRP3 (ab263899, Abcam), SOCS3 (bs-24250R, Bioss), TSG101 (bs-1365R, Bioss), CD63 (bs-23032R, Bioss), calnexin (bsm-52639R, Bioss), and β-actin (bs-0061R, Bioss) at 4 °C overnight, followed by application with Goat Anti-Rabbit IgG (ab672, Abcam) for 1 h. The membranes were developed using an enhanced chemiluminescence solution (Yeasen), and the band intensity was quantified by Image J software.