Hrp conjugated a nipure goat anti rabbit igg h l

Western blotting was performed as described previously [19] (link). Blotting bands were then incubated with primary antibodies overnight at 4°C. Antibodies against the following proteins were used: c-PARP (Cell Signaling Technology #5625, 1:1000), PROCA1 (Biorbyt #orb1703, 1:1000), ZFP36L2 (Santa Cruz #sc-365908, 1:1000), c-Myc (Cell Signaling Technology #9402, 1:1000), phosphor-AKT (Cell Signaling Technology #4060, 1:1000), AKT (Cell Signaling Technology #4691, 1:1000), phosphor-PI3K (Cell Signaling Technology #17366, 1:1000), and PI3K (Cell Signaling Technology #4292, 1:1000). Internal reference antibodies were used: GAPDH (Cell Signaling Technology #5174, 1:1000), β-Tubulin (Proteintech #10068-1-AP, 1:1000). Secondary antibodies used as follows: HRP-conjugated A nipure Goat Anti-Rabbit IgG(H+L) (Proteintech #SA00001-2, 1:5000) and HRP-conjugated A nipure Goat Anti-Mouse IgG(H+L) (Proteintech #SA00001-1, 1:5000) were purchased from Proteintech (Rosemont, IL, USA). Images were captured by chemiluminescence (Bio-rad, Hercules, California) and quantitated using a Quantity One system (Bio-Rad, Hercules, CA, USA).

Total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% protease inhibitors and phosphatase inhibitors. Protein concentration was measured with the bicinchoninic acid (BCA) assay. Western blot analysis was performed as previously described(16). BRIX1 (sc-373680, Santa Cruz), GLUT1 (ab115730, Abcam), β-catenin (ab32572, Abcam), Ki67 (Proteintech Group, Inc.) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated A nipure Goat Anti-Rabbit IgG (H+L) and HRP-conjugated A nipure Goat Anti-Mouse IgG (H+L) were obtained from Proteintech Group, Inc (Jackson).