Figure 8.

p62 depletion decreases the insoluble ubiquitinated (Ub−) proteins and the insoluble CryAB^R120G protein in NRVMs overexpressing CryAB^R120G (R120G) and exacerbates cell injury, and reduces cell survival in cultured NRVMs overexpressing CryAB^R120G or MT-des. NRVMs were treated as described in Figure 6. A ~ D, Cells were lysed with a lysis buffer containing 1% Triton X-100. Triton X-100 soluble and insoluble fractions of the cell lysates were used for western blot analysis for the abundance of ubiquitinated proteins or HA-tagged CryAB^R120G. GAPDH and α-actinin were probed as loading controls for proteins in the soluble and the insoluble fractions, respectively. Representative images (A) and a summary of quantitative data (B, C and D) are presented. E ~ G, The following assays were performed on day 7. E, LDH activities in the cultured medium were measured to assess cell injury. F, Cell viability was assessed by the MTT assay. Cell viability was deceased by expressing CryAB^R120G or MT-des, and was further reduced by p62 knockdown. G, Changes in the LDH/MTT ratio. * p<0.05, vs. β-gal/luc-siRNA; # p<0.05; n=4.