Antibodies against hif 1α

BMS202 (PD-1 inhibitor), SC79 (Akt activator), and NSC87877 (SHP1/2 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, and IL-1β were purchased from Elabscience Biotechnology (Wuhan, China). Antibodies against HIF-1α, SHP1, SHP2, phosphorylated Akt (p-Akt), Akt, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against PD-1, CD11c, CD16, CD86, and CD86/PE were from Bioss (Beijing, China). Fluorophore-labeled goat anti-rabbit secondary antibody (Alexa Fluor 594), goat anti-rabbit immunoglobulin horseradish peroxidase (IgG-HRP), bovine serum albumin (BSA), and the bicinchoninic acid (BCA)-based protein assay were purchased from Beyotime (Shanghai, China). The 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System kit was from Solarbio Life Sciences (Beijing, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA); PrimeScript™ RT Kit, from Takara (Osaka, Japan); fetal bovine serum (FBS); and Ham’s F-12 K (Kaighn’s) medium, from Gibco (Carlsbad, CA, USA).

Cells were cross-linked with 1% formaldehyde and subsequently immunoprecipitated with antibodies against HIF1α (Cell Signaling, Danvers, MA, USA). The PCR primers were designed to amplify the promoter regions containing HRE within miR-519d promoter region. A negative control with IgG was used. Fold enrichment was calculated as a ratio of amplification efficiency of the ChIP sample over that of IgG.

The detailed procedure for western blotting has been described [44 (link)]. Western blotting used primary antibodies (diluted 1:1000) against the following proteins: AKT (sc-8312), phosphorylated AKT (sc-16646-R), ERK (sc-94), phosphorylated ERK (sc-7383), and vimentin (sc-6260); each antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against HIF-1α (Cell Signaling) and E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ) were also used. Proteins separated by SDS-PAGE were transferred to a Hybond-C Extra membrane (GE Healthcare, Little Chalfont, UK) that was then subjected to western blotting with an appropriate primary antibody. Anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an ECL western blot detection system (Millipore, Bedford, MA, USA), and band intensities were quantified by densitometry (Digital Protein DNA Imagineware, Huntington Station, NY).