Vps34

Western blotting analysis of proteins in flap tissue was conducted as described previously (Chen et al., 2020 (link)), including the following proteins: VEGF (1:1,000, ProteinTech), Cadherin 5 (1:1,000, Affinity), LC3II (1:500, ProteinTech), SOD1 (1:1,000, ProteinTech), C-CASP3 (1:1,000, CST), MMP9 (1:1,000, Abcam), SQSTM1/p62 (1:1,000, Abcam), Bax (1:1,000, ProteinTech), CTSD (1:1,000, ABclonal), HO-1 (1:1,000, ProteinTech), Bcl-2 (1:1,000, CST), eNOS (1:1,000, Abcam), VPS34 (1:1,000, ProteinTech), Beclin1 (1:1,000, ProteinTech), PI3K (1:1,000, ProteinTech), AKT (1:1,000, ProteinTech), p-PI3K (1:1,000, CST), p-Akt (1:1,000, CST), AMPK (1:1,000, CST), p-AMPK (1:1,000, CST), HIF-1α (1:1,000, Abcam), and GAPDH (1:1,000, ProteinTech). Finally, the intensity of Western blotting was quantified using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).

Protein was extracted from lung tissues on ice for 30 min using RIPA lysis buffer with proteinase and phosphatase inhibitors (100:1:1). The tissue lysate was then centrifuged for 30 min at 13,000 rpm and 4°C. Using bovine serum albumin as a reference, protein content was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). 30 μg total proteins of lung tissue lysate were run on 10% tris-glycine SDS-PAGE gel (Biosharp), 80 V for 0.5 h and 110 V for 1 h. Gels were transferred to a polyvinylidene difluoride (PVDF) membrane by Trans-Blot (Bio-Rad, USA), and blocked with 5% BSA for 1 h. The bands were incubated overnight at 4°C with primary antibodies: PI3K (1:1,000, CST, USA), p-PI3K (1:1,000, CST, USA), AKT (1:1,000, CST, USA), p-AKT (1:2000, CST, USA), mTOR (1:1,000, CST, USA), p-mTOR (1:1,000, CST, USA), Beclin-1 (1:1,000, CST, USA), Atg5 (1:1,000, CST, USA), LC3B (1:1,000, CST, USA), VPS34 (1:500, Proteintech, China), β-actin (1:20000, Proteintech, China). Proteins on the band were observed using a ChemiDoc XRS Gel Imaging System (Bio-Rad, USA) after 1 h at room temperature incubation with the appropriate secondary antibodies (Fcmacs, China). ImageJ software was used to examine the protein expression of each group (v1.8.0).