Protein expression levels were assessed by immunoblot analysis of cell lysates (20–50 μg protein) in RIPA buffer containing 1× phosphate-buffered saline (PBS), 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, and protease inhibitors. Primary antibodies to CD133 (11,000, Proteintech, CA, USA), SOX2 (1:1000, Proteintech), Nanog (1:1000, Proteintech), β-Actin (1500, Zsbio, Beijing, CHN), PRRX1 (11,000, Abcam, USA), STAT3 (11,000, Affinity, UK), p-STAT3 (11,000, Affinity), and CXCR4 (11,000, Proteintech) were used.
For co-IP assays, cells were transiently or stably transfected with the indicated constructs. The cells were harvested and lysed in 1 ml of lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). The resulting lysates were subjected to immunoprecipitation with antibodies directed to the epitope tag. Immunoprecipitates were washed in lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis, and subsequently analysed by protein immunoblotting.
For co-IP assays, cells were transiently or stably transfected with the indicated constructs. The cells were harvested and lysed in 1 ml of lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). The resulting lysates were subjected to immunoprecipitation with antibodies directed to the epitope tag. Immunoprecipitates were washed in lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis, and subsequently analysed by protein immunoblotting.