Ab55906

Whole protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime) and quantified using the bicinchoninic acid Protein Assay Kit (Beyotime). Briefly, a 20 μg protein aliquot was loaded into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation, followed by electroblotting on a polyvinylidene fluoride membrane (Merck Millipore, Burlington, MA, USA), blocking using 5% fat-free milk, and overnight incubation with the following primary antibodies: anti-UBR5 (ab70311; Abcam), anti-AXIN1 (ab55906; Abcam), anti-β-catenin (ab32572; Abcam), anti-Survivin (ab76424; Abcam), anti-C-myc (ab39688; Abcam), anti-lactate dehydrogenase A (LDHA, ab101562; Abcam), anti-pyruvate kinase isozymes M2 (PKM2, ab137852; Abcam), anti-H3 (ab1791; Abcam), and anti-GAPDH antibody (60004-1-1G; ProteinTech, Rosemont, IL, USA). They were washed thrice with Tris-buffered saline with Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies (A0208, A0216; Beyotime). Finally, the blot was imaged using the Enhanced Chemiluminescence Detection Kit (Pierce Biotechnology), and the band intensity was measured using Image-Pro Plus 6.0 software, with GAPDH as the loading control.

Cells were harvested, washed twice by ice-cold PBS, and subjected to lysis. Proteins (20 µg) from cell lysates were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk and immunoblotted with primary antibodies (rabbit anti-mouse Axin1 (ab55906, Abcam), rabbit anti-mouse Wnt1 (ab85060, Abcam), rabbit anti-mouse β-catenin (ab196204, Abcam)) overnight at 4°C followed by incubation at room temperature with a peroxide-conjugated secondary antibody (Beyotime, China). Proteins were then visualized by an enhanced chemiluminescence method (Beyotime, China) according to the manufacturer’s instructions.