Fig. 4.

MEST interacts with VCP and accelerates IκBα degradation. A H1299 cells were transfected with MEST-FLAG expression plasmids, and MEST-binding proteins were identified by co-immunoprecipitation coupled with LC/MS analyses. A list of candidate proteins was uploaded for Ingenuity Pathway Analysis, which revealed that the top canonical pathways involved membrane trafficking. B Silver staining proteins coimmunoprecipitated with MEST show a specific 97 kDa band corresponding to VCP. C H1299 cells were transfected with MEST-FLAG and VCP-HA expression plasmids. The interaction between MEST and VCP was demonstrated by co-immunoprecipitation assays. D A549 cells transfected with MEST-eGFP (green) were stained with VCP antibody (red) for 48 h, and co-localization of MEST and VCP in the perinuclear area was imaged by using a confocal microscope; intensity spatial profiles are plotted. Scale bar, 20 μm. E Fluorescence resonance energy transfer experiment showed the energy transfers from MEST-eGFP (donor) to VCP-mCherry (acceptor). Expression of an eGFP-mCherry fusion protein is a positive control, and co-expression of eGFP-N1 and mCherry-N1 is a negative control. Excitation wavelength is 488 nm, and collection wavelength is 600 nm. Scale bar, 20 μm. F Schematics for the structural domains of VCP and for the N-terminal deletion (ΔN) and C-terminal deletion (ΔC) mutants. The blue line represents the AAA domain of VCP. G The interactions of VCP-truncation mutants (WT, ΔN, and ΔC) with MEST were detected by co-immunoprecipitation assay. H MEST increases the binding of VCP to p-IκBα. H1299 cells were transfected either with or without MEST-FLAG expression plasmids for 48 h; cell lysates were incubated with anti-VCP antibody, and expression of p-IκBα was assessed by western blot analysis. I Bay11-7082 abolishes interactions between MEST, VCP, and IκBα. A549 cells expressing Myc-tagged IκBα were treated with Bay11-7082 (2.5 μM) for 12 h. Cell lysates were incubated with anti-Myc antibody for co-immunoprecipitation assay, and VCP and MEST were detected by western blot analysis. J MEST-overexpressing H1299 cells, MEST-overexpressing A549 cells, and their respective vector control cells were treated with cycloheximide (CHX; 50 μg/mL). The cell lysates were collected at the indicated time points and compared for IκBα expression by western blot analysis. IκBα signals are quantified by densitometry, and the degradation rate is shown as the ratio of IκBα expression level at each time point to the original expression level (0 h). The half-life (t_1/2) of IκBα was 0.9 and 3.6 h in MEST-overexpressing H1299 cells and corresponding vector control cells, respectively; t_1/2 values were 1.8 and 28.6 h in MEST-overexpressing A549 cells and vector control cells, respectively