Figure 4.

Role of the ubiquitin/proteasome pathway in the posttranslational regulation of Ha-Ras and Ki-Ras by hydrogen peroxide. Immunoblot analysis of Ha-Ras and Ki-Ras in pan-Ras immunoprecipitates in cultures pretreated for 15 min with the proteasomal inhibitor, MG-132 (40 μM), and then challenged with hydrogen peroxide for 5 min is shown in (a). Values are expressed as per cent of controls and represent the means + SEM of 3 determinations. Note the lack of additivity between MG-132 and hydrogen peroxide in increasing Ha-Ras and Ki-Ras levels. Ubiquitin immunoreactivity in pan-Ras immunoprecipitates from cultures treated with MG-132 and/or hydrogen peroxide is shown in (b). Note that a 5-min treatment with hydrogen peroxide reduces protein ubiquitination both in the absence and presence of MG-132. The temporal profile of Ha-Ras and Ki-Ras expression in cultures pretreated with MG-132 for 15 min and then challenged with hydrogen peroxide for 30 and 60 min is shown in (c). Expression after a 5-min exposure with hydrogen peroxide alone is also shown for comparison. Values are expressed as per cent of controls and are means ± SEM of 3 determinations. Note that pretreatment with MG-132 did not affect the late reduction in Ha-Ras and Ki-Ras expression.