FIGURE 1:

Characterization of gp78-knockout MEFs. (A) Schematic of gp78 gene-targeting strategy. The wild-type allele of murine gp78 contains 14 exons. Excision of the sequences between the loxP sites deletes exons 7 and 8, which encode part of the most C-terminal transmembrane region and the adjacent RING finger domain of gp78. (B) Representative PCR analysis of genomic DNA from tails of wild-type (WT), heterozygous (HET), and gp78^−/− (knockout [KO]) embryos. (C) Levels of gp78 protein in primary MEFs derived from gp78 WT, HET, or KO embryos were assessed by immunoblotting using gp78 Ab2. β-Actin was used as a control for equal loading. (D) To rule out the possibility that gp78^−/− MEFs express a truncated form of gp78 terminating before exon 7 (see Material and Methods), levels of gp78 in primary MEFs from KO or WT embryos from the same litter were probed with affinity-purified rabbit antiserum directed against an N-terminal transmembrane region of mouse gp78 (aa 33–54; gp78 Ab3), which is encoded by exon 1. Arrow indicates the expected migration of truncated gp78 fragment if present. Results from two different sets of primary MEFs are shown. (E) Paired gp78 KO and WT MEFs were treated with 50 μg/ml cycloheximide (CHX) to inhibit protein synthesis, and degradation of CD82/KAI1 was monitored by immunoblotting with an antiserum recognizing murine CD82 (MK-35). Calnexin is shown as a control for equal loading.