FIGURE 2.
Analysis of HRD1-SEL1L complex by sucrose density gradient centrifugation. HEK293 cells were treated with 30 nm of StealthTM siRNA of negative control (Control) or targeted for SEL1L (siSEL1L), HRD1 (siHRD1), or OS-9 (siOS-9) for 48 h and were extracted by a buffer containing 3% digitonin. After fractionation by 10–40% sucrose density gradient centrifugation with a 60% sucrose cushion, SEL1L (A), HRD1 (B), and OS-9 (C) were detected by Western blotting. Solid bars indicate the fractions where the large ERAD complex containing HRD1-SEL1L and OS-9 sedimented. The dotted line shows the fractions of OS-9 with lower sedimentation rates. Asterisks indicate the nonspecific signals detected by the mouse monoclonal anti-SEL1L antibody. Open and gray arrowheads indicate the positions of OS-9v1 and v2, respectively.