Anti taz

Sections of 5 μm thickness were dewaxed in xylene and rehydrated in serial dilutions of ethanol. Antigen retrieval was carried out in 0.01 M sodium citrate buffer (pH 6.0) for 10 min by microwave oven heating. Then endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 20 min, and nonspecific staining was blocked by 5% BSA for 1 h. The slides were incubated with anti-TAZ (1:100, Santa Cruz Biotechnology) and anti-PCNA (1:1000, Santa Cruz Biotechnology) overnight at 4°C and then incubated with secondary antibody for 30 min, followed by incubation with streptavidin peroxidase for 15 min. 3,3′-Diaminobenzidine tetrachloride (DAB) was applied to identify peroxidase activity. Each incubation step was performed at room temperature and was followed by sequential washed for 3 min each for three times in PBS. Finally, sections were dehydrated in alcohol and cleared in xylene.

To evaluate related protein level after treatment, HNSCC cells were collected and lysed by RIPA lysis buffer. Proteins with equal concentration were subjected to the SDS-PAGE and transferred to PVDF membrane (BIO RAD, USA). All samples were blocked by nonfat milk (5%) and incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-EMP1 (Santa Cruz Biotechnology, CA, USA), anti-YAP (Cat, #ab52771), anti-TAZ (Cat, #560235), anti-nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX4) (Cat, #ab133303), anti-acyl-CoA synthetase 4 (ACSL4) (Cat, #ab155282), anti-GPX4 antibody (Cat. #ab125066), anti-Rac1 (Cat. #ab33186), anti-GAPDH (CST, #5176), and anti-nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) (Cat, #ab55831). Antibodies for phospho-epidermal growth factor receptor (EGFR) and EGFR were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-AK, AKT, Phospho-ERK1/2, and ERK1/2 were from Cell Signaling Technology (Beverly, MA). On the next day, all membranes were incubated with the secondary antibody incubation for 2 hours at room temperature. The expression result was detected by ECL reagent (Sigma, USA, WBULS0100). Per condition analyzed a minimum of 10,000 cells.

Erlotinib (Santa Cruz) was dissolved in Dimethy1 Sulfoxide (DMSO). Mouse monoclonal anti-TAZ, anti-cyclin E2, anti-CDK2 and anti-GAPDH were obtained from Santa Cruz. Rabbit monoclonal anti-EGFR, anti-phospho-EGFR, anti-AKT, anti-phospho-EGFR, anti-ERK1/2, anti-phospho-ERK1/2 and anti-p21 were obtain from Cell Signaling Technology. Mouse monoclonal anti-Ki67 and propidium iodide (PI) were obtained from BD Biosciences.