Persistent oxidative stress promotes proteasome-dependent degradation of H2AX mediated by RNF168 and ubiquitination of K119.Figure 3A Up, Representative western blot from chromatin-free fraction or whole cell extracts, as indicated, showing H2AX protein levels in wt and junD-/- fibroblasts. Different amounts of loaded proteins and exposures (low- or high-exp) are presented, H2AX being easier to detect when associated with chromatin. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper). B Up, Representative western blot from chromatin-free fraction extracts showing H2AX protein levels in wt and junD-/- fibroblasts either untreated (-) or after 8h treatment (+) with proteasome inhibitor MG132. Jun is used as positive control for treatment efficiency. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).C Bar plot showing stabilization of H2AX protein in wt and junD-/- fibroblasts, as assessed by densitrometry analysis of western blots (as shown in B). Data are expressed as percentage (%) of H2AX protein levels after treatment with MG132 with respect to untreated conditions. D Representative western blots from whole cell extracts showing HA-tagged ubiquitin (Left) or Flag-tagged H2AX (Right) protein levels. wt and junD-/- fibroblasts were co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were then immunoprecipitated with Flag-specific antibody and incubated either with HA-specific antibody (Left) or Flag-specific antibody (Right). Two different exposures (low or high exp) are presented. * stands for the immunoglobulins. E Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels (revealed using Flag-specific antibody) from whole cell extracts in wt and junD-/- fibroblasts. Cells were first transfected with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and next treated with cycloheximide (CHX) for the indicated times (hours, h). Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in wt and junD-/- fibroblasts, as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part. F Left, Representative western blots showing H2AX-WT protein levels (revealed using Flag-specific antibody) from whole cell extracts in junD-/- fibroblasts co-transfected with Flag-tagged H2AX-WT construct and either non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), RNF8 (siRNF8) or BMI1 (siBMI1). Efficiency of each siRNA has been verified and reached 60% of inhibition, in average, as shown for RNF168 (Up). Protein extracts were processed following cycloheximide (CHX) treatment for the indicated times (hours, h). Right, Bar graph showing the percentage (%) of decrease of H2AX-WT protein levels after 4h treatment with CHX (t=4) with respect to untreated conditions (t=0) in junD-/- fibroblasts transfected with specific siRNA, as indicated. G Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels from whole cell extracts in junD-/- fibroblasts transfected either with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and co-transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168). Cells were treated with cycloheximide (CHX) for the indicated times (hours, h) before protein extraction. Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in junD-/- fibroblasts transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part. H Representative western blot showing RNF168, Flag-tagged H2AX (H2AX-FLAG) and endogenous H2AX (H2AX) protein levels from whole cell extracts in wt and junD-/- fibroblasts. Cells were first co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were next immunoprecipitated with Flag-specific antibody and incubated either with RNF168-specific antibody (Up) or H2AX-specific antibody (Down).