Ab129473

Wild-type control or BTK-knockdown (shBTK) OSCC tumorspheres were plated in six-well chamber slides for 24 h for immunofluorescence analysis. The tumorspheres were fixed with 2% paraformaldehyde and probed with primary antibodies against BTK, TIM-3, CD133, Krüppel-like factor 4 (KLF4) (1:500, ab129473, rabbit; Abcam), E-cadherin (24E10) (1:500, #3195, rabbit mAb; Cell Signaling), and vimentin. A fluorophore-conjugated secondary antibody was added to check the positive signal using a Zeiss Axiophot (Carl Zeiss) fluorescence microscope. The nuclei of viable cells were detected through 4′,6-diamidino-2-phenylindole (DAPI) staining.

Total protein of midbrain tissues and cells was obtained using Invitrogen Lysis buffer and quantified by the BCA method (Thermo Fisher Scientific). An equal amount of protein (50 μg) was subjected to electrophoresis on 10% SDS-polyacrylamide gels and then blotted on nitrocellulose membranes (Thermo Fisher Scientific). After being blocked in 5% non-fat milk, the membranes were probed with anti-KLF4 (ab129473; Abcam) or anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, ab9485; Abcam) antibody, followed by the addition of horseradish peroxidase-conjugated secondary antibody (ab205718; Abcam). Immunoblots were developed by the enhanced chemiluminescence method (Thermo Fisher Scientific) as recommended by the manufacturers.

Cells were harvested in cold PBS and lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris at pH 7.5, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 10 mM EDTA) containing protein inhibitor cocktail (Roche, Basel, Switzerland). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibodies and HRP-conjugated secondary antibody. β-Actin was used as a loading control. The antibodies used in this study were as follows: human anti-SHCBP1 (1:400; SAB1307183, Sigma-Aldrich, USA), anti-MYC (1:5,000; ab32072, abcam, USA), anti-EGFR (1:1,000; ab52894, abcam, USA), anti-KLF4 (1:1,000; ab129473, abcam, USA), anti-CD44 (1:1,000; ab51037, abcam, USA), anti-VEGFA (1:1,000; ab46154, abcam, USA), anti-CDK4 (1:500; 11026-1-AP, Proteintech, USA), anti-CCND1 (1:500; sc-8396, Santa Cruz Biotechnology, USA), anti-ITGA6 (1:400; sc-19622, Santa Cruz Biotechnology, USA), anti-ITGB1 (1:1,000; ab52971, abcam, USA), anti-ITGB5 (1:1,000; ab15459, abcam, USA), anti-COL4A2 (1:2,000; 55131-1-AP, Proteintech, USA), anti-GAPDH (1:2,000; sc-32233, Santa Cruz Biotechnology, USA), and HRP-conjugated secondary antibody (1:5,000; G-21040, Thermo Scientific, USA). The signals were detected by the Li-Cor Odyssey system (Li-Cor Biosciences, Lincoln, NE, USA).