Phosphorylated mtor p mtor

Western blotting was conducted following the protocol outlined by Song et al. (2018), with minor modifications [43 (link)]. The primary antibodies used in this study were interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p-STAT3), AKT serine/threonine kinase 1 (Akt1), mTOR, phosphorylated mTOR (p-mTOR), nuclear factor (NF)-κB, MMP9 (all at a dilution of 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:500; Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin antibody (1:10,000; Cell Signaling Technology). A horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G secondary antibody (1:5,000; GenDEPOT, Katy, TX, USA) was used to visualize the blots. The blots were visualized using West-Q Pico Dura and Femto ECL solution (GenDEPOT).

Anti-mouse and anti-rabbit horseradish peroxidase-conjugated IgG, rabbit polyclonal antibodies against AdipoR1, AdipoR2, β-actin, phosphorylated-p85 (p-p85), p85, phosphorylated-Akt (p-Akt), Akt, phosphorylated-mTOR (p-mTOR), and mTOR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HIF-1α, VEGF-A, and CD31 antibodies were purchased from Abcam (Cambridge, MA, USA). Recombinant human adiponectin and VEGF-A were purchased from R&D Systems (Minneapolis, MN, USA). pHRE-luciferase construct was provided from Dr. W.M. Fu (National Taiwan University, Taiwan). The pSV-β-galactosidase vector and luciferase assay kit were purchased from Promega (Madison, WI, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).