Fig. 2.

DRiPs interact with the HB complex to activate the response to proteasome failure. (A) Incubation with the proline analog AZC enhances the effects of proteasome inhibition. MCF10A cells were left untreated or were incubated for 3 h with the indicated concentrations of MG132 alone or combined with 2.5 mM AZC. YM1, an inhibitor of the HB complex, reverses the effects of proteasome inhibition on LATS1/2 activity. Cells were preincubated with YM1 for 3 h and were incubated with 5 μM MG132 combined with 5 μM YM1. (B) Slowing down translation lessens the effects of proteasome inhibition on the suppression of LATS1/2. MCF10A cells were left untreated or were incubated for 3 h with 1 μM MG132 alone or combined with 0.15 μM emetine. Results of an analogous experiment with the addition of cycloheximide are shown in SI Appendix, Fig. S2A. (C) The effects of proteasome inhibition, inhibition of translation, and disruption of the HB complex on the association of Hsp70 and LATS1 with ubiquitinated species. MCF10A cells were incubated for 2 h with the indicated combinations of 2.5 μM MG132, 0.5 μM emetine, or 10 μM YM1. All cells incubated with YM1 were preincubated for 1.5 h. After the treatments, cells were incubated for 10 min with 1.2% formaldehyde to cross-link protein complexes. Ubiquitinated species were isolated from the cell lysates and analyzed by immunoblotting. To measure Hsp70, we used a mixture of antibodies against HspA1 and HspA8. Typical results of two independent experiments are shown.