Bs1511

Western blotting was performed with the SDS-PAGE electrophoresis system. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The following primary antibodies were used: anti-GAPDH (ap0063, Bioworld), anti-Col15α1 (ab150463, Abcam), anti-Caspase-9 (ab32539, Abcam), anti-Cleaved Caspase-9 (bs7070, Bioworld), anti-Caspase-3 (Bs6428, Bioworld), anti-Cleaved Caspase-3 (bs7004, Bioworld), anti-Bcl2 (bs1511, Bioworld), anti-Bax (ab32503, Abcam), anti-AMPK (ab32047, Abcam), anti-pAMPK (ab133448, Abcam), anti-mTOR (ab87540, Abcam), anti-pmTORC1^Ser2448 (ab109268, Abcam), anti-Akt (ab8805, Abcam), anti-pAkt^Ser473 (ab18206, Abcam), anti-S6K1 (ab32529, Abcam), anti-pERK1/2 (ab201015, Abcam), anti-ERK1/2 (ab17942, Abcam), anti-pS6K1^Thr389 (ab2571, Abcam), anti-Collagen I (ab34710, Abcam), anti-Collagen VI (ab6588, Abcam), anti-Fibronectin (ab2413, Abcam), anti-MMP2 (ab92536, Abcam), anti-MMP9 (ab38898, Abcam), anti-TIMP1 (WL02342, Wanleibio), anti-TIMP2 (ab180630, Abcam), anti-FGFR1 (ab31324, Abcam), anti-pFGFR1^{Tyr653/Tyr654} (GTX133526, GeneTex), anti-TGFβ1 (WL03092, Wanleibio). Horseradish peroxidase anti-rabbit or anti-goat (Sigma–Aldrich) were used as secondary antibodies.

SGC-7901 cells were prepared as the Con, P and G groups. At the end of the experiments, total cellular protein was extracted from tissue specimens and gastric cancer cells using a lysis buffer containing 1X Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentration was quantified using the Bicinchoninic Protein Assay kit (KeyGEN, China). Equal quantities of protein samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and electroblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked in 5% non-fat milk overnight, and the following day, membranes were incubated with a rabbit polyclonal anti-GDDR (ab70480, Abcam, Cambridge, UK), rabbit monoclonal anti-Fas (5709-1, Epitomics, Inc., CA, USA), anti-bcl-2 (BS1511, Bioworld, St. Louis, MN, USA), anti-Bax (BS2538 Bioworld) or anti-GAPDH (BSAP0063 Bioworld) for 4 h. Following washing with phosphate-buffered saline (PBS) with Tween-20 four times, and incubation with goat anti-rabbit secondary antibody (GAR0072, LiankeBio, Hangzhou, China) for 2 h at room temperature, the protein bands were visualized using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA).

Protein from white adipocytes or white adipose tissue was extracted using lysing buffer. Protein concentration was determined using BCA Protein Assay kit (Beyotime, Nanjing, China). The experimental procedure was as described previously [46 (link)]. Briefly, Protein samples (30 μg) were separated by SDS-PAGE and transferred to PVDF nitrocellulose membranes (Millipore, USA), and blocked with 5% Skim Milk Powder/Tween 20/TBST at room temperature for 2 h. Then, primary antibodies against Bcl-2 (bs1511), Cleaved Caspase-9 (bs7070), Cleaved Caspase-3 (bs7004), Gapdh (ap0063), β-actin (ap0060) (Bioworld, CA, USA), Hoxa5 (ab140636), Bax (ab32503), Cytochrome C (ab53056), Akt (ab8805), p-Akt^Ser473 (ab81283), mTOR (ab87540), p-mTORC1^Ser2448(ab137133), and S6K1 (ab9366), p-S6K1^Thr389 (ab2571) (Abcam, Cambridge, UK) were used to incubate the membranes at 4 °C overnight and the appropriate HRP-conjugated secondary antibodies (Boaoshen, China) were used for 2 h at room temperature. Akt phosphorylation-specific inhibitor MK2206 and mTORC1 inhibitor rapamycin were purchased from Selleck Chemical (USA). Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, USA).