Figure 8

Effects of Cav-3 WT or Cav-3 V82I expression on ERK1/2 activation. BHK cells were transiently transfected with Cav-3 WT or Cav-3 V82I. (a) Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting using anti-ERK and anti-pERK antibodies. Expression of total cellular level of ERK remained unaltered after the transfection with either Cav-3 WT or Cav-3 V82I in all the conditions tested. On the contrary, a significant decrease in pERK (active form) was observed in the presence of the caveolin-3 mutant, both under resting conditions and after 30 min hyperosmotic stress with mannitol. (b) Quantization revealed that in Cav-3 V82I expressing cells there was a significant decrease of ERK activation as compared with Cav-3 WT and mock transfected cells. Data are representative of three independent experiments. **, P < 0.01 vs the respective mock and Cav-3 WT groups. Similar results were obtained in (c,d). Cell lysates from control and mannitol treated groups were subjected to an in vitro ERK1/2 kinase assay as described in Materials and Methods. Representative blot is shown in (c) and relative band intensities of pElk analysis is shown in (d). Data are representative of three independent cultures. *, P < 0.05 V82I vs WT in untreated cells; **, P < 0.01 WT vs all groups.