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. 2014 Jun 9;9(6):e92164. doi: 10.1371/journal.pone.0092164

Figure 1. ERAD components required for the degradation of N278A.

Figure 1

(A) N278A-HA was stably expressed in 293T cells. Protein synthesis was inhibited with cycloheximide (CHX), and the fate of the protein was followed. Samples were analyzed by SDS-PAGE and immunoblotting with HA antibodies. Immunoblotting with p97 and G3PDH were used as loading controls. (B) Cells stably expressing N278A-HA were depleted of AAA-ATPase p97, and the fate of N278A proteins was followed after CHX addition. All samples were analyzed by SDS-PAGE and immunoblotting with HA antibodies. The extent of depletion was shown by immunoblotting with p97 antibodies. Controls were treated with commercial negative control siRNA. The right graph shows quantification of N278A in the experiment. Immunoblotting for GAPDH served as loading control. (C) As in (B), but with depletion of ubiquitin ligase HRD1. Immunoblotting for p97 served as loading control. (D) As in (C), but with depletion of SEL1L. Molecular masses are given in kilodaltons.