Mapk6

After operation, paraffin embedded LSCC tissue and adjacent tissue sections (4 μm) were removed and dewaxed with xylene, absolute ethanol, phosphate buffered saline (PBS), and 3% hydrogen peroxide was used to inactivate endogenous peroxidase 19 (link). Then the sections were removed and washed with PBS. The antigen was repaired by thermal repair method. And then the sections were removed and blocked for 10 min. The specific primary antibody was added and incubated at 4°C for 12 h. Specific primary antibodies were as follows: ZNF671 (Sigma-Aldrich, Shanghai, China; HPA046099, 1:100), MAPK6 (Abcam, Cambridge, MA, USA; ab53277, 1:100), Ki67 (Abcam, ab15580, 1:100), E-cadherin (Abcam, ab76055, 1:100), N-cadherin (Abcam, ab76011, 1:100). After incubation, the cells were washed, and then biotin-labeled secondary antibody and horseradish peroxidase labeled chain enzyme ovalbumin were added successively, and the cells were incubated at 4°C for 20 min. After incubation, the sections were washed again and stained with chromodevelopment solution for 5 min. After washing, counterstaining, dehydration and sealing, the sections were placed under a microscope for observation. Five high-power fields were randomly selected from each section and the number of positive staining cells was counted. The reagents used in this experiment were purchased from Wuhan Goodbio Technology Company.

The paraffin-embedded tissue sections were subjected to dewaxing, rehydration, as well as antigen repair. Next, the tissue sections were incubated at 4 °C overnight with Ki67 (1:100, Cell Signaling Technology), MAPK6 (1:50, Abcam) and Caspase-7-specific antibody (1:500, Abcam) followed by 1-h incubation at 37 °C with the biotin-labeled secondary antibody. Lastly, the tissues were stained with DAB and hematoxylin.

Total proteins from BC cells were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer containing phenylmethyl sulfonylfluoride (PMSF), followed by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After that, the PVDF membranes were blocked with 5% skimmed milk and incubated with the corresponding primary antibody: Bax (1:1000, Abcam, Cambridge, UK), Bcl-2 (1:1000, Abcam), MAPK6 (1:1000, Abcam), p-MAPK6 (1:1000, Abcam), p38 (1:1000, Cell Signaling Technology, Danvers, MA, USA), p-p38 (1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), and β-actin (1:5000, Cell Signaling Technology). Next, the membranes were incubated at 4℃ for 14 h, and subsequently, the membranes were incubated with the corresponding secondary antibody (1:6000, Cell Signaling Technology) for 1.5 h at room temperature. Lastly, the band signal was visualized using an enhanced chemiluminescence (ECL) detection system (Millipore).