FIGURE 3.

The ubiquitous 180-kDa JLP is specifically depleted in cells by siRNA. A and B, a CHO-based TRVb1/TTF cell line (A) or mouse 3T3L1 adipocytes (B) were transfected with siRNA duplexes targeting mouse JLP or control cyclophilin B as detailed under “Experimental Procedures.” Seventy-two h post-transfection, cell lysates (140 μg of protein) were analyzed by SDS-PAGE and immunoblotting (IB) with anti-JLP antiserum. Shown is a relatively high exposure blot, so that the selective ablation of endogenous JLP (lane 1 versus 2 in A and B, arrowheads) and absence of an off-target effect are apparent. C, COS7 cells were transiently transfected with pCMV5-Myc-mJLP_L^WT cDNA or the empty vector as indicated. Lysates were collected 24 h post-transfection. Equal protein amounts (60 μg) were analyzed by SDS-PAGE and immunoblotting with anti-Myc antibodies. Depicted is the 180-kDa Myc-JLP_L^WT. A–C, shown are chemiluminescence detections of representative immunoblots from two to five independent experiments with similar results.