Akt1 2 3

Protein was extracted from tissue with RIPA buffer (Tris-HCl (pH 7.4) 50 mM, NaCl 150 mM, NP-40 1%, Sodium deoxycholate 0.5%, SDS 0.1%, EDTA 1 mM). For immunoblotting assay, 40 μg protein samples were separated by SDS-PAGE, and detected with antibody against Gab2 (Cell Signaling, 3239), p-Gab2(Tyr452) (Cell signaling, 3881), Ucp1 (Abcam, ab10983), p-Akt(Ser473) (Cell Signaling, 4060), Akt(1/2/3) (Santa Cruz, 8312), p-Erk1/2(Thr202/Tyr204) (Cell Signaling, 9101), Erk (Cell Signaling, 9102), p-Stat3(Tyr705) (Cell Signaling, 9131), Stat3 (Cell Signaling, 9139), β-Tubulin (TransGen, HC101-01), PI3K p85 (Cell Signaling, 4257), p-FoXO1(Thr24)/3a(Thr32) (Cell Signaling, 9464), FoXO1 (Cell Signaling, 2880).
For coimmunoprecipitation analysis, adipose tissues or cell samples were lysed with lysis buffer (Tris-HCl (pH 7.5) 20 mM, NaCl 150 mM, TritonX-100 1%, glycerinum 5%, Sodium deoxycholate 1%, EDTA 5 mM, phosphatase inhibitor, protease inhibitor). After centrifuging (13,500 rpm, 4 °C, 15 min), lysates were applied for immunoprecipitation with antibodies coated protein G agarose beads at 4 °C overnight. Then the beads were washed with lysis buffer five times (30 s), and the immunoprecipitated results were analyzed by immunoblotting.

Protein samples were extracted from whole heart homogenates (n = 6/group, chosen randomly) and ADSCs as previously described [21 (link)]. Primary antibody against tumour necrosis factor-α (TNF-α) (R&D, Minneapolis, MN, USA), Akt1/2/3 (Santa Cruze, Santa Cruz, CA, USA), phospho-Akt (Santa Cruze), ERK (Cell Signaling), phosphor-ERK (Cell Signaling), signal transducers and activators of transcription 3 (STAT3; Cell Signaling), phosphor- STAT3 (Cell Signaling), Bcl-2 (Santa Cruze), Bax (Santa Cruze) and horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) were used according to manufacturers’ instructions. GAPDH and β-actin antibody (Cell Signaling Technology) was used to evaluate the amount of protein loaded in each sample (detailed in the supplementary material).

Cell extracts were separated by SDS-PAGE and proteins identified by western blotting, as previously described.^{15 (link)} Antibodies used for western blot detection were as follows: Axl (C-20), pEGFR, Akt 1/2/3 and pAkt 1/2/3 (Santa Cruz), EGFR (Cell Signalling Technology, Leiden, The Netherlands) and pAxl 779 (Bio-techne). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit (Dako, Cambridge, UK), anti-goat and anti-mouse Igs (Promega, Southampton, UK).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Millipore Sigma (Billerica, MA, USA). Recombinant human TNF-α and recombinant human IFN-γ were purchased from Bio-Techne Ltd. (Abingdon, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Primary antibodies against p-IKK α/β (cat no. 2697), NF-κB p65 (cat no. 8242), p-Akt (cat no. 9271), and ICAM-1 (cat no. 4915) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against IKK α/β (cat no. 7607), p-IκB-α (cat no. 8404), IκB-α (cat no. 203), Akt1/2/3 (cat no. sc-8312), PARP (cat no. sc-9542), α-tubulin (cat no. sc-8035), Filaggrin (cat no. sc-66192), VCAM-1 (cat no. sc-1504), E-selectin (cat no. sc-5262), and β-actin (cat no. sc-81178) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories, Inc. (West Grove, PA, USA). The histamine ELISA kit was obtained from Enzo life Sciences, Inc. (Farmingdale, NY, USA). The ELISA kits for TNF-α and IL-6 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).