Alexa fluor 594 conjugated goat anti mouse 488 conjugated goat anti rabbit secondary antibodies

A549 cells were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized by 0.1% Triton X-100 in PBS, blocked with 4% bovine serum albumin (BSA), and incubated with primary antibody (dilution ratios: phospho-p38 MAPK—1 : 500, phospho-p65—1 : 500, p38 MAPK—1 : 1000 dilution, p65—1 : 1000, and FKN—1 : 1000) overnight at 4°C. Cells were washed and incubated with Alexa Fluor® 594-conjugated goat anti-mouse/488-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 50 min in the dark. Cells were washed three times, mounted with propidium iodide- (PI-) containing mounting media (ZSGB-BIO, Beijing, China), and visualized using a confocal laser scanning microscope. FKN-positive cell numbers were counted in every 300 cells, and then the FKN-positive cell ratio was calculated.

MRC-5 cells seeded on glass slides were fixed in 90 % cold ethanol, permeabilized by 0.1 % Triton X100 in PBS, blocked with 5 % bovine serum albumin (BSA), and incubated with anti-PTEN (26H9, #9556, Cell Signaling Technology, Danvers, MA, USA) or anti-USP13 (ab99421, Abcam) overnight at 4 °C. Cells were washed and incubated with Alexa Fluor® 594-conjugated goat anti-mouse/488-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 50 min in the dark. Cells were washed three times, mounted with 4'6-diamidino-2-phenylindole (DAPI)-containing mounting media (ZSGB-BIO, Beijing, China), and visualized using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Hesse, Germany).