Usp40

The antibodies listed below were utilized: USP40 (cat# sc-514248, Santa Cruz), USP40 (ab121234, Abcam), Claudin1 (cat# 13255, Cell Signaling Technology), Claudin1 (cat# sc-166338, Santa Cruz), Claudin1 (cat# 13050-1-AP, Proteintech), HA-tag (cat# 51064-2-AP, Proteintech), c-Myc (ab32072, Abcam), KLF4 (cat# 12173, Cell Signaling Technology), Ub (cat# sc-8017, Santa Cruz), and β-actin (cat# 66009-1-Ig, Proteintech). Cycloheximide (CHX, cat# S7418) and MG132 (cat# S2619) were purchased from Selleck.

Cells were fixed with PHEMO buffer (0.025 M HEPES, 0.068 M PIPES, 0.003 M MgCl₂·6H₂O, 0.015 M EGTA·Na₂, 10% DMSO, pH adjusted to 6.8. Additional reagents were added before use, with a final concentration as follows: 0.05% glutaraldehyde, 0.5% Triton X-100, 3.7% formaldehyde) for 10 min at room temperature before washing with PBS for 3 times. Then, cells were incubated with blocking buffer (3% BSA) for 30 min at room temperature. Afterward, cells were incubated with primary antibodies against CFLAR_L (Santa Cruz, dilution at 1:500) for 1 h at room temperature. After washing with PBS for 3 times, cells were incubated with another primary antibodies GMEB1 (Santa Cruz, dilution at 1:500) or USP40 (Santa Cruz, dilution at 1:500) for 1 h. Alex Fluor 488 (Green) and Alex Fluor 568 (Red)-conjugated secondary antibodies were then applied and incubated at room temperature for 1 h. Cell nuclei were stained with DAPI. Images were captured using a confocal microscope (ZEISS, LSM700).