ERAD components required for the degradation of HShh-C. (A) Cells were depleted of the ER luminal lectin OS9 by siRNA, and the fate of stably expressed HShh-HA was followed after cycloheximide (CHX) addition. The extent of OS9 depletion (in parentheses) was determined by quantitative RT-PCR. Controls were treated with an unrelated siRNA. All samples were analyzed by SDS-PAGE and immunoblotting with HA antibodies. The right graph shows quantification of HShh-C in the experiment. All samples were also analyzed by immunoblotting for p97 (loading control). (B) As in A, but with depletion of the ER luminal lectin XTP3 by two different siRNAs. (C) As in A, but with depletion of the ubiquitin ligase Hrd1 by two different siRNAs. (D) As in A, but with depletion of the Hrd1-interacting protein Sel1 by two different siRNAs. (A–D) n = 3 time points. (E) As in A, but with depletion of the ATPase p97 by siRNA. n = 4 time points. Molecular masses are given in kilodaltons.