Skip to main content
. 2015 Oct 25;7(7):7640–7656. doi: 10.18632/oncotarget.6156

Figure 1. MNT as a novel interacting partner of E6AP.

Figure 1

a. GST and GST-E6AP proteins were expressed in E. coli with 0.2mM IPTG induction for 4h at 37°C and 16h at 20°C respectively, isolated and purified with glutathione sepharose 4B beads b. Colloidal coomassie stained gel showing GST pull down performed using GST and GST-E6AP from WCEs of 1.0μM ATRA treated HL60 cells. c. WCEs of 1.0μM ATRA treated HL60 cells for 0, 8, 12 and 24h were resolved on 8% SDS-PAGE and immunoblotted with MNT, E6AP and β-actin antibodies. d. GST pull-down with GST and GST-E6AP from lysates of 293T transfected with 0.5ug HA-MNT was performed. e. Co-immunoprecipitation with E6AP antibody was performed from lysates of HEK293T co-transfected with 1.0μg HA-MNT and 0.5μg E6AP as indicated. The blot was probed with anti-E6AP followed by anti-HA antibodies. Cells were treated with 10μM MG132 and 10μM Lactacystin (LCT) 6h prior to harvesting, as indicated. f. 293T cells were co-transfected with 0.5μg HA-MNT and 0.5μg E6AP. IFC was performed 24h post-transfection in order to avoid heavier degradation of MNT. We followed a regular IFC protocol and incubated cells overnight with ant-E6AP and anti-HA antibodies. Next day, cells were incubated with anti-rabbit (Alexafluor 594) and anti-mouse (Alexafluor 488) secondary antibodies. Cells were dried and mounted to capture the images; 10μM MG132 treatment was given 6h prior to fixing for immunostaining. g. HL60 cells were treated with 1μM ATRA for 24h and cells were treated with 10μM MG132 6h prior to harvesting as indicated. Co-immunoprecipitation using MNT antibody was performed and immunoblotted with anti-MNT followed by anti-E6AP antibody after stripping the same blot; * indicates unstripped MNT from upper panel. h. Endogenous E6AP was co-immunoprecipitated using E6AP antibody from U937 WCEs treated with 5nM PMA for 24h. Co-immunoprecipitates were resolved and probed with E6AP followed by MNT antibody. Cells were treated with 10μM MG132 6h prior to harvesting as indicated. Results are representative of minimum three independent experiments.