Figure 2.

BiP is required for binding of ERdj5 but not ERdj4 to misfolded SP-C. (A) Cell lines stably expressing WT or SP-C^Δexon4 (x4) were transfected with plasmids encoding BiP and treated with MG-132; no cross-linking agent was added in this experiment. Cell lysates were immunoprecipitated with SP-C proprotein antibody followed by Western blotting with FLAG antibody to detect BiP. EV, HEK293 cells transfected with empty vector (pIRES2-EGFP). (B) a, HEK293 cells were cotransfected with plasmids expressing ERdj4 or ERdj4^ΔJ and BiP. After cross-linking, cell lysates were immunoprecipitated with HA antibody and analyzed by Western blotting with FLAG antibody to detect BiP or HA antibody to detect ERdj4. b, HEK293 cells stably expressing SP-C^WT (lanes 1) or SP-C^Δexon4 (lanes 2) were transfected with plasmids encoding ERdj4^ΔJ, treated with MG-132, and cross-linked. Cell lysates were immunoprecipitated with SP-C antibody and analyzed by Western blotting with HA antibody to detect ERdj4^ΔJ. (C) a, HEK293 cells were cotransfected with plasmids encoding ERdj5 or ERdj5^ΔJ and BiP. After cross-linking with DSP, cell lysates were immunoprecipitated with FLAG antibody (BiP) and analyzed by Western blotting with HA antibody to detect ERdj5 or FLAG antibody to detect BiP. b, HEK293 cells stably expressing SP-C^WT (lanes 1) or SP-C^Δexon4 (lanes 2) were transfected with plasmids encoding ERdj5^ΔJ, treated with MG-132, and cross-linked. Cell lysates were immunoprecipitated with SP-C antibody and analyzed by Western blotting with HA antibody to detect ERdj5^ΔJ. (D) HEK293 cells stably expressing SP-C^WT or SP-C^Δexon4 were transfected with plasmids encoding ERdj4 or ERdj4^H54Q. Forty-eight hours after transfection, cells were pulse-labeled with [³⁵S]Met/Cys for 15 min, and then they were chased for the indicated times. After cross-linking with DSP, cell lysates were immunoprecipitated with HA antibody followed by reducing SDS-PAGE, and autoradiography.