Ab18206

Male C57BL6/J mice weighing 20–25 g were housed at the Ethics Committee for Animal Experiments of the First Hospital of China Medical University under a 12 h:12 h light/dark cycle with a constant temperature (22 ± 1 °C) and free access to food and water. All animal experiments were performed according to the National Institutes of Health guide for the care and use of Laboratory animals, and were approved by the Animal Care and Use Committee of the First Hospital of China Medical University.
The detailed information of antibodies are as follows: Anti-ILEI antibody (ab72182, Abcam, Cambridge, U.K.), Anti-α‐SMA antibody (ab5694, Abcam), Anti-vimentin antibody (ab137321, Abcam), Anti-E‐cadherin antibody (ab133597, Abcam), Anti-p‐Akt antibody (ab18206, Abcam), Anti-Akt antibody (ab126811, Abcam), Anti-p‐ERK antibody (ab223500, Abcam), Anti- ERK antibody (ab17942, Abcam), Anti-collagen Ι antibody (ab34710, Abcam), Anti-LIFR antibody (sc-659, Santa cruz, Dallas, U.S.A.),Anti-collagen ΙII antibody (WL03186,wanleibio,Shenyang, China), Anti-β‐actin antibody (WL01845,wanleibio).

The protein contents of vascular endothelial growth factor A (VEGFA), PI3K, p-PI3K, Akt and p-Akt were evaluated by western blot and the detailed protocol as described previously (Song et al. 2019 ). In brief, 20 µg of protein isolated from cells were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and membrane transfer, followed by blocking in 5% non-fat milk. After that, the membranes were incubated with primary antibodies at 4 °C overnight, and then incubated with horseradish peroxidase (HRP)-conjugated IgG (ab205718; 1:10,000; Abcam, Cambridge, MA, USA) for 2 h at room temperature. The antibodies were as follows: anti-VEGFA (ab214424; 1:1000; Abcam), anti-cleaved caspase-3 (ab184787; 1:1000, Abcam), anti-PI3K (ab154598; 1:1000; Abcam), anti-p-PI3K (#4228; 1:600; Cell Signalling Technology, Boston, MA, USA), anti-Akt (ab8805; 1:1000; Abcam), anti-p-Akt (ab18206; 1:1000; Abcam) and anti-β-actin (ab8226; 1:1000; Abcam). Enhanced chemiluminescence reagent (ECL; Sigma) was applied to detect the protein bands. For in vitro cell samples, tests were performed with three biological replicates and three technical replicates. For animal tissue samples, tests were performed with six biological replicates and three technical replicates.

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.