FIGURE 4.

Association of the ERAD substrate to XTP3-B requires mannose trimming, even after knockdown of SEL1L. A, plasmids encoding for S-tagged XTP3-B and H2a cDNAs were cotransfected into HEK-293 cells with or without ERManI shRNA in a pSUPER plasmid. 24 h after transfection, cells were incubated for 5 h in the absence/presence of 100 μm Kif. Cells were lysed in 1% Nonidet P-40, 50 mm Tris/HCl (pH 8), 150 mm NaCl and 10% of the lysates were run on 10% SDS-PAGE and immunoblotted with anti-S tag antibodies (bottom panel). The rest of the lysates were immunoprecipitated (IP) with anti-H2a and protein A-Sepharose. Eluted samples were subjected to 10% SDS-PAGE and immunoblotted with anti-S tag (upper panel) or anti-H2a (middle panel). Results were quantitated and normalized as in Fig. 2A; values are averages of three independent experiments, error bars are S.D.; p < 0.04 for all coimmunoprecipitations compared with the control. B, experiment similar to that in A, except for SEL1L shRNA that was used instead of ERManI shRNA (p = 0.03). C, in parallel with B, HEK-293 cells were transfected with the plasmid encoding anti-SEL1L shRNA used in B or with pSUPER encoding anti-LacZ shRNA (control, cont). RNA was extracted 24 h post-transfection and used for RT-PCR with primers for SEL1L mRNA (upper panel) compared with GAPDH (lower panel).