Mapk8

The cells transfected with different siRNAs were harvested following 24 hours of transfection and washed once with cold PBS. Cells were then suspended in an appropriate volume of lysis buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA,1 mM EGTA, 1 mM PMSF and 1% Triton X-100) and incubated for 30 minutes at 4 °C. Cells were then centrifuged at the highest speed for 15 minutes and the supernatant was collected for quantification using the BCA protein quantification kit (Pierce, USA). 20 μg of proteins were separated by 4–12%% SDSPAGE and then transferred onto nitrocellulose membranes (Amersham Pharmacia, UK). Rabl3 antibody (Proteintech, USA, 1:1000), LC3 antibody (Cell Signaling, USA, 1:1000), MAPK8 (Cell Signaling, USA, 1:2000), MAPK14 (Proteintech, USA, 1:2000), p-MAPK8/9/10 (Cell Signaling, USA, 1:1000) and p-MAPK11/12/13/14 (Cell Signaling, USA, 1:1000) and Actin Beta Antibody (Sigma, USA, 1:5000) were used.