Anti ddx5

For immunohistochemistry (IHC), human colorectal cancer tissue microarray slides (Outdo Biotech, Shanghai, China, #HColA180Su08). Patient information provided by the TMA supplier. After the tissue sections were deparaffinized and rehydrated, antigen retrieval was carried out in citrate buffer (Beyotime) at 100°C for 0.5 hour. Endogenous peroxidase was blocked with 3% peroxide for 15 minutes and blocked in buffer (5% BSA +0.1% Triton X‐100) for 1 hour and incubated overnight in primary antibody. The primary antibodies used were anti‐DDX5 (Santa Cruz, #sc‐166167, 1:300), anti‐O‐GlcNAcylation (Abeam, #ab2739, 1:100), and detection of signals using a Vectastain ABC kit (Vector Labs, Burlingame, CA, USA). For IF, cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS and blocking buffer (3% FBS +1% HISS +0.1% Triton X‐100), and then incubated overnight at 4°C in primary antibody. The primary antibodies used were anti‐DDX5 (Santa Cruz, #sc‐166167, 1:100), anti‐O‐GlcNAcylation (Abeam, #ab2739, 1:100). Fluorescent Alexa‐Fluor‐488 or ‐555‐conjugated secondary antibodies (Life technologies, Carlsbad, CA, USA) were used for detection.

For Western blot (WB), the protein was resolved on SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel followed by standard WB. The primary antibodies used were anti‐GAPDH (CST, #5174, 1:2000), anti‐β‐Actin (CST, #3700, 1:2000), anti‐OGT (Abcam, #ab184198, 1:1000), anti‐Flag tag (Sigam,#F2555), 1:1000), anti‐DDX5 (Santa Cruz, #sc‐166167, 1:1000), anti‐O‐GlcNAcylation (Abcam, #ab2739, 1:1000), anti‐AKT (CST, #4691, 1:1000), anti‐p‐AKT (CST, #4060, 1:1000), anti‐mTOR (CST, #2972, 1:1000), and anti‐p‐mTOR (CST, #5536, 1:1000). Observe proteins with enhanced chemiluminescence reagents.