Goat anti mouse igg

Total protein was isolated using RIPA buffer (Applygen, Beijing, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland) according to the manufacturers’ instructions. Western blot was performed by routine operation. Immunoblotting was carried out with primary antibodies against FKBP10 (1:500, 50353, Sigma), Hsp47 (1:500, sc-5293, Santa Cruz), p-AKT (Ser473) (1:1000, 4060S, CST), Akt (1:1000, 2920S, CST), p-CREB (1:1000, 9198S, CST), CREB (1:1000, 9197S, CST), PCNA (1:1000, 13110S, CST). GAPDH (1:5000, 60004-1-Ig, 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).

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).

Proteins were extracted from cells by RIPA buffer (1% Triton X‐100, 0.1% sodium dodecyl sulphate, 1% sodium deoxycholate, 0.15 m NaCl and 10 mm Tris, pH 7.2) with a protease inhibitor cocktail Set III (539134, Merck). Western blotting was performed according to the standard procedures. The polyvinylidene fluoride (PVDF) membrane was incubated in primary antibodies CDHR5 (SC‐166953, Santa Cruz; 1:1000), GAPDH (H‐12, Santa Cruz; 1:1000) or β‐actin (4970S, Cell Signaling Technology; 1:1000) overnight at 4°C and then in secondary antibodies goat antimouse IgG or goat anti‐rabbit IgG (C1308 and C1309, Applygen; 1:10 000) at room temperature for about 1 hour. Western blot signals were detected by using Immobilon Western Chemiluminescent HRP Substrate (WBKLS0050).