Fig. 4

USUV NS4A is targeted by p62 for autophagic degradation. A 293T cells were co-transfected with expression plasmids for HA-tagged USUV NS4A and FLAG-tagged p62. At 24 hpt lysates were collected and immunoprecipitated with a FLAG antibody and the whole cell lysates and immunoprecipitates were analysed for the indicated proteins by western blot. B 293T cells were transfected with the USUV NS4A expression plasmid. 24 hpt cells were treated with 30 µg/mL cycloheximide (CHX) and with DMSO or 100nM bafilomycin A1 (BafA1). At the indicated times cells were harvested for immunoblotting with HA and GAPDH. Graph below the blots shows the band intensity ratio of HA to GAPDH and are normalized to the untreated cells (0 h). C 293T cells were transfected with the USUV NS4A expression plasmid and increasing amounts of the p62 plasmid (0.5 µg, 1 µg, and 1.5 µg). At 24 hpt cells were harvested and immunoblotted for HA, FLAG, and GAPDH. D 293T cells were co-transfected with the USUV NS4A and p62 expression plasmids. At 24 hpt cells were treated with 30 µg/mL CHX for the indicated times and harvested for immunoblotting with HA, FLAG, and GAPDH. Graph on the right shows the band intensity ratio of HA to GAPDH and are normalized to the untreated cells (0 h). E 293T cells were transfected with siRNA targeting p62 or the non-targeting pool (NTP). 48 h after the siRNA transfection, cells were transfected with the USUV NS4A expression plasmid. At 24 hpt cells were treated with 30 µg/mL CHX for the indicated times and harvested for immunoblotting with HA, p62, and GAPDH. Graph on the right shows the band intensity ratio of HA to GAPDH and are normalized to the untreated cells (0 h). F 293T cells were transfected with the USUV NS4A expression plasmid and plasmids expressing either the wildtype p62 (wt) or a mutant p62 lacking the LIR motif (ΔLIR). At 24 hpt cells were harvested and immunoblotted for HA, FLAG, and GAPDH. Numbers between the panels represent the band intensity ratio of HA to GAPDH and are normalized to the NS4A-transfected cells