FIGURE 5.
The E3 ligases HRD1 and SCFFbs2 are involved in ERAD of H2a. HEK 293 cells stably expressing H2aSBP were transiently transfected with plasmids encoding dominant negative mutant Myc-tagged HRD1 (mut HRD1) or FLAG-tagged ΔF box-Fbs2 (Fbs2ΔF). Two days after transfection, cells were labeled with [35S]Cys, and each pool of labeled cells was divided into aliquots that were subjected to 0 or 5 h chase. At the end of the pulse or chase periods, the cells were lysed and immunoprecipitated with anti-H2a. The eluted samples were subjected to SDS-PAGE followed by phosphorimaging detection and quantification. Note that H2aSBP is constructed with an uncleavable H2a variant that does not yield fragment. The plotted data provide quantification analysis of the stability of H2a upon coexpression of Fbs2ΔF or mutant HRD1 (average of three independent experiments); p < 0.03 for expression of both dominant negative proteins compared with control.