FIGURE 5.

HERPs link DERL2 to HRD1-SEL1L complex. A, 293T cells were depleted of HERP1 or/and HERP2 by siRNA. Cells were treated with cycloheximide and lysed in lysis buffer containing 1% Triton X-100 and protease inhibitors. Cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with indicated antibodies. Immunoblotting with GAPDH was used as loading control. The depletion was also quantified by quantitative RT-PCR (in parentheses). B, as in A, but with cells stably expressing SHH-HA depleted of SEL1L, HERP1, and HERP2 as indicated. Immunoblotting with p97 was used as loading control. C, cells stably expressing HRD1-Myc were treated with chemical cross-linkers dithiobis(succinimidyl propionate) (DSP) as described under “Experimental Procedures.” Cell lysates were then immunoprecipitated (IP) with Myc antibodies. Immunoblotting with HRD1 antibodies was carried out. D, as in C, but with cells depleted of both HERP1 and HERP2. Immunoblotting was carried out with the indicated antibodies. E, 293T cells were depleted of HERP1, HERP2, or both HERPs. Cells were lysed in HKM buffer with 1% deoxy-bigChap and protease inhibitors and immunoprecipitated with HRD1 antibodies. The precipitates and lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. CHX, cycloheximide.