Ab187524

Whole-cell protein and tissue protein extraction was isolated as described previously [33 (link)]. Western blotting were performed as previously described [33 (link)]. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millopore) and exposure to X-Omat film (Kodak). Anti-hCCL28, anti-mCCL28 (Santa Cruz, sc-27341), anti-mVEGFa (Abcam, ab51745), anti-hHIF1α (Abcam, ab92498), and anti-mHIF1α (Abcam, ab187524).

The collected cells were rinsed in cold PBS three times. Then, 100~200 μL of RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was added to lyse the cells in ice-cold water under ultrasound, and the Bradford method was used to check the protein concentration. Proteins of identical amounts were taken from each group for 10% SDS-PAGE electrophoresis, and the proteins on the gel were transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After being sealed for an hour at 4 °C, the membranes were incubated with anti-TRIM14 (15742-1-AP, 1:500, Proteintech, Wuhan, China), anti-STAT3 (ab68153, 1:1000, Abcam, Cambridge, UK), anti-p-STAT3 (phospho Y705, ab76315, Abcam, 1:1000), anti-HIF-1α (ab187524, Abcam, 1:1000), anti-p62 (ab109012, Abcam, 1:1000), anti-LC3B (ab63817, Abcam, 1:2000), anti-Beclin1 (ab207612, Abcam, 1:2000), anti-E-cadherin (ab76319, Abcam, 1:1000), anti-N-cadherin (ab76011, Abcam, 1:2000), anti-Snail (ab180714, Abcam, 1:1000), anti-Vimentin (ab92547, Abcam, 1:1000), and anti-β-actin (ab8226, 1:000, Abcam) overnight at 4 °C. Then, goat anti-rabbit IgG H&L (HRP) (ab97051, 1:1000) was administered for 1 h of hybridization at indoor temperature. The membranes were flushed with TBST three times for 5 min each. The ECL chemiluminescence substrate was used for color development, and X-ray films were exposed for 30 s to 5 min.