Bs7070

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.

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).