Immunofluorescence

MDA-MB-231 cells transfected with an empty vector or a TIPE1 overexpression vector were dispensed onto gelatinized slides in 24-well plates at a density of 5,000 cells per well. Then, the cells were fixed for 20 min with 4% formaldehyde solution and permeabilized for 5 min with 0.5% Triton X-100 solution. After washing twice (2 × 10 min in PBS), slides were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline containing 0.2% Tween-20, incubated overnight with the anti-p-ERK antibody [1:500 dilution; #4370 (Thr202/Tyr204), CST, USA] at 4°C, washed (2 × 10 min in PBS), incubated with an Alexa Fluor 594-labeled secondary antibody (Thermo Fisher) for an hour at room temperature, and then washed twice with PBS. A coverslip was then mounted onto the slides with Prolong Gold Antifade reagent with DAPI (Life Technologies) and washed twice (2 × 10 min in PBS). The fluorescence images of the cells were acquired with a fluorescence microscope equipped with appropriate filter combinations.