P anxa2 tyr23

The cells that grew on the slides were fixed, permeabilized, blocked and incubated with MYC (1:100) (Abcam), p-ANXA2 (Tyr23, 1:100) (Santa Cruz) or His-tag (1:100) (Proteintech) at 4 °C overnight. The bound primary antibodies were detected using goat anti-mouse IgG-FITC or goat anti-rabbit IgG H&L (1:100) (Abcam) at 37 °C for 1 h. The fluorescence was detected via confocal microscopy (General Electric Company, Fairfield, CT, USA).

Total protein was isolated using RIPA buffer (Applygen, Beijing, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Nuclear and cytoplasmic protein was isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Immunoblotting was performed with primary antibodies against ANXA2 (1:200), p-ANXA2 (Tyr23, 1:200), VEGF (1:200) (Santa Cruz Biotechnology, Dallas, Texas, USA), MYC (1:1000), p-SRC (Tyr418, 1:1000) (Abcam, Cambridge, UK), Ubiquitin (1:500), Histone H3 (1:1000) (CST, Danvers, MA, USA), HIF1A (1:500), SRC (1:500), HA-tag (1:3000), His-tag (1:3000) (Proteintech, Wuhan, China). GAPDH (1:500) (Proteintech) was used as a loading control. Secondary antibodies (Goat anti-Mouse IgG and Goat anti-Rabbit IgG, 1:5000) were purchased from Applygen. The signals were visualized with a super enhanced chemiluminescence (ECL) detection reagent (Applygen). Quantitative analysis of immunoblotting was performed using ImageJ (Ver. 1.52a, NIH image, Bethesda, MD, USA).