Mouse Anti-human CD24 antibody (Cat.: ab30350), rabbit anti-human CD44 antibody (Cat.: ab157107), rabbit anti-human CD133 antibody (Cat.: ab19898), rabbit anti-human Oct4 antibody (Cat.: ab19857), rabbit anti-human Nanog antibody (Cat.: ab21624), rabbit anti-IRE1 (phosphor S724) antibody (Cat.: EPR5253), rabbit anti-IRE1 antibody (Cat.: ab96481), rabbit anti-PERK (phosphor T982) antibody (Cat.: ab192591) and rabbit anti-PERK antibody (Cat.: ab65142) were obtained from Abcam (Cambridge, England) and diluted followed the manufacturer’s instructions.
Total protein was prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) followed the manufacturer’s instruction. The same amount of protein from total protein was fractionated using Tris-glycine gels, and transferred to PVDF membranes. After transferring, PVDF membranes were blocked in 5% milk/TBS buffer at room temperature for 30 min, and this was followed by an incubation with primary antibodies at 4°C overnight. After washing with PBS-T (containing 0.1% Tween-20) for three times, HRP-conjugated secondary antibodies were incubated with PVDF membranes for another 1 hour. Blots were developed using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
Total protein was prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) followed the manufacturer’s instruction. The same amount of protein from total protein was fractionated using Tris-glycine gels, and transferred to PVDF membranes. After transferring, PVDF membranes were blocked in 5% milk/TBS buffer at room temperature for 30 min, and this was followed by an incubation with primary antibodies at 4°C overnight. After washing with PBS-T (containing 0.1% Tween-20) for three times, HRP-conjugated secondary antibodies were incubated with PVDF membranes for another 1 hour. Blots were developed using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.