Ha ha 7 h9658

Cells were plated on coverslips and transfected following overnight incubation. Cells were fixed with 3% paraformaldehyde, permeabilized in 0.5% Triton-X100 for 10 min and blocked in 5% FBS diluted in PBS. Coverslips were incubated overnight with primary antibody HA (HA-7, H9658, Sigma-Aldrich) or FLAG (M2, F1804, Sigma-Aldrich), washed in PBS and incubated with anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Life Technologies, Burlington, ON, Canada). Cells were mounted with ProLong Gold antifade with DAPI (Invitrogen). Visualization was done using an Olympus BX51 microscope with a 40x objective and images were captured with the Image-Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA). For quantification analysis, images were blinded by a third party.

Equal amounts of protein from cell lysates were separated by SDS PAGE and transferred onto a nitrocellulose membrane by a semi-dry transfer method (Trans Blot^® SD Semi-Dry Transfer cell, Bio-Rad, USA). Membranes were blocked with 5 % milk solution before incubation with primary antibodies at 1:1000 dilution unless otherwise stated: USP13 (D4P3M; 12577, CST), Mcl-1 (S-19; sc-819, SCBT), Phospho-c-Jun (Ser73) (D47G9; 3270, CST), c-Jun (60A8; 9165, CST), FLAG (F1804, Sigma-Aldrich), HA (HA-7; H9658, Sigma) and GAPDH (SCBT; sc365062) (1:5000) as a loading control. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma Aldrich, USA) were used at a 1:5000 dilution. Proteins were detected using WesternBright ECL (Advansta, USA) and visualised on X-ray film.