FIGURE 4.

ERdj4 is not integrated in the ER membrane. A, fractionation of endogenous ERdj4 from P3U.1 cells. The membranes were prepared from 2.5 × 10⁷ cells and divided evenly into five aliquots. After pelleting by centrifugation, the membranes were resuspended in either PBS or PBS containing 0.1% digitonin, 1% deoxycholate (DOC), or 0.1 m Na₂CO₃. The samples were rocked at 4 °C for 1 h and then centrifuged to pellet the membranes. Pellets (P) and supernatants (S) were subjected to electrophoresis and immunoblotting with the indicated antibodies. B, COS-1 cells were transfected for 24 h with pSGmERdj4. The membranes were processed, separated by gel electrophoresis, and immunoblotted with the indicated antibodies as in A.