Ab216647

Total proteins from different groups of PTC cells were extracted by RIPA lysis buffer (Beyotime) and qualified by a BCA detection kit (Sigma). Subsequently, proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride (PVDF, Millipore) membranes. Primary antibodies suck as rabbit anti‐ALDH1 (ab52492, 1:500, Abcam), rabbit anti‐CD44 (ab216647, 1:1000, Abcam), rabbit anti‐NANOG (ab109250, 1:500, Abcam), rabbit anti‐SOX2 (ab97959, 1:500, Abcam), rabbit anti‐Oct4 (ab18976, 1:1000, Abcam), rabbit anti‐TLR4 (ab13556, 1:1000, Abcam), and β‐actin (ab8226, 1:1000, Abcam) were incubated with membranes at 4°C overnight after blocked in 5% nonfat milk. On the next day, the membranes were washed and then incubated with Goat Anti‐Rabbit secondary detection antibodies (1:4000; ab205718; Abcam). The protein of interest was detected using the LI‐COR Odyssey infrared imaging system (LI‐COR Bioscience).

Total cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific), separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked using fat-free milk and incubated with specific antibodies at 4 °C overnight. The primary antibodies were all purchased from Abcam (Cambridge, MA, USA), including anti-Bax (ab32503), anti-Bcl-2 (ab185002), anti-MMP2 (ab215986), anti-CD44 (ab216647), anti-SOX2 (ab97959), anti-Oct4 (ab93689), anti-Nanog (ab218524), anti-β-catenin (ab32572), anti-cyclin D1 (ab1663), anti-c-myc (ab32072), and anti-GAPDH (ab8245). Membranes were then cultivated with secondary antibody (ab205718; Abcam) and detected by chemiluminescence system (Bio-Rad).