Figure 4. Proteasome inhibition modifies the relative quantity of Hsf2α and Hsf2β at the mRNA level.

(A) WT iMEFs were treated with MG132 at 1 µM or with DMSO for 10 h, or were heat shocked 20 min at 45°C and allowed to recover during 6 h. The relative quantity of Hsf2α and Hsf2β transcripts was assessed by RT-PCR using primers flanking the alternative exon. Gapdh RT-PCR served as control for efficient retrotranscritpion and amplification. (B) WT iMEFs were treated during 2 h, 4 h, 6 h or 10 h with MG132 at 1 µM or with DMSO before being harvested. In addition, WT iMEFs exposed to MG132 during 10 h were allowed to recover during 1 h, 6 h or 24 h before collection. Relative quantity of Hsf2α and Hsf2β was assessed by RT-PCR, as described above. Quantity one software (Bio Rad) was used to assess band intensity. Results were expressed in mean of percentage of each isoform +/− SD from four independent experiments. (C) WT iMEFs treated as described in (B) were harvested for protein analysis. Protein extracts were loaded on 12% polyacrylamide gel and submitted to a long migration to separate efficiently HSF2 isoforms. HSF2 was revealed by immunoblotting. Ponceau staining was used to verify the equal loading.