Hy n0901

At 20 h after transfection, HEK293T cells transfected with pcDNA3.1, HA-REEP6, HA-REEP6 c.268G>C, and HA-REEP6 c.468delC were treated with 100 μM cycloheximide (CHX, HY-N0901, MedChemExpress, NJ, USA) dissolved in DMSO. Cells were scraped at 0, 7, and 10 h after CHX exposure using ice-cold PBS. Cells were centrifuged, and lysed by RIPA lysis buffer (PC101, EpiZyme, Shanghai, China) including 1% protease inhibitor cocktail (GRF101, EpiZyme). After brief sonication, cell lysate was collected. The protein concentration was determined with BCA protein assay kit (P0011, Beyotime, Shanghai, China). All samples were boiled for 5 min at 100 °C, diluted by 5X SDS loading buffer (LT101S, EpiZyme). Equal amounts of protein were loaded on a 12.5% polyacrylamide gel for SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore, Burlington, MA, USA). PVDF membranes were blocked in 5% skim milk prior to incubation with mouse anti-HA (1:500; 2367S, Cell Signaling Technology, Beverly, MA, USA) and rabbit anti-β-actin (1:1000; 4970S, Cell Signaling Technology). After 2 h of incubation with secondary antibodies at room temperature, the membranes were visualized on a Bio-Rad ChemiDoc MP Imaging System. β-Actin was used as loading control.

After 24 h cell culture, HEK293T cells transfected with pcDNA3.1, Flag-CFAP410, Flag-CFAP410 c.319 T > C, and Flag-CFAP410 c.347C > T were treated with 100 μM cycloheximide (CHX, HY-N0901, MedChemExpress, NJ, United States) or CHX mixed inhibitors of proteasome (MG132). Cells were scraped at 0, 6, 12, and 18 h after CHX/MG132 exposure using ice-cold PBS. The total protein was extracted for Western blot. Wild-type and mutant CFAP410 protein levels were detected using flag antibody. β-actin were the loading control (32 (link)).