Goat anti rabbit igg

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).

Plant samples (200 mg) of roots, stems, and leaves were lysed by using Radio Immunoprecipitation Assay (RIPA lysis buffer) following protocol (Beyotime Institute of Biotechnology) and homogenized in the ice-cold RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0, and a mixture of protease inhibitors (Applygen Technologies Inc.)). After the precipitate was discarded, crude membrane fraction was collected by centrifugation at 12,000 RPM for 20 min at 4°C. Protein samples (30 µg) were separated by 12% SDS-PAGE gels and then blotted onto PVDF membrane (EMD Millipore) following the standard protocol. Primary antibody (anti-LRB7) was used to detect TIP, followed by secondary antibody (goat-anti-rabbit IgG, Applygen Technologies Inc.). The signals were detected and visualized by using ECL Western Blotting system (Amersham Bioscience).

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).