(A) Yeast two-hybrid analyses of the p97-Rep8 interaction using the HIS3 reporter gene. Co-transformation of bait p97 with prey Rep8 supported cell growth under conditions selecting for the interaction (in the absence of histidine and presence of 25 mM 3-aminotriazol (3AT); right panel). The known p97-p47 interaction served as a control. (B) Schematic diagram of the Rep8 domain organization and the various truncations used in the precipitation experiments. (C) Purified p97 was incubated with the indicated, immobilized GST fusion proteins before analysis of bound proteins by SDS-PAGE and blotting using antibodies specific for p97 (top panel) or GST (lower panel).

(A) Schematic diagram of the p97 domain organization and the various truncations used in the precipitation experiments. (B) Purified 6His-tagged p97 and p97 truncations were incubated with immobilized GST or GST-tagged Rep8 before analysis of the bound proteins by SDS-PAGE and blotting using antibodies specific for the 6His-tagged p97 proteins (top panel) or GST (lower panel). The p97 input has been included for comparison. (C) Endogenous p97 was immunoprecipitated from HeLa cell lysates. The precipitated material was analyzed by blotting using antibodies to p97 and Rep8. (D) Endogenous p97 was immunoprecipitated from rat testis homogenate. The precipitated material was analyzed by blotting using antibodies to p97 and Rep8.

(A) The insoluble fraction of a HeLa cells lysate was isolated by centrifugation. Pellets were mixed with sucrose, 0.5 M NaCl, 100 mM Na₂CO₃ or 0.1% SDS as indicated, and separated by centrifugation into a pellet (P) and supernatant fraction (S) prior to analysis by SDS-PAGE and blotting using antibodies to p97, calnexin, actin and Rep8. To ease comparison, the pellets were resuspended in the same volumes as the supernatant prior to analysis. All Rep8 was insoluble and only released with SDS, but not with sucrose, sodium chloride or sodium carbonate. (B) Confocal micrographs of formaldehyde-fixed HeLa cells transfected to express Rep8 with a C-terminal GFP-tag (left panel) and RFP modified to contain a signal sequence and an ER-retention signal (ER-targeted RFP; middle panel). In the merged image (right panel), the signals overlap (yellow). (C) Differential centrifugation of HeLa cells transfected to express Rep8-GFP as in (A). TMX3 and the proteasome subunit Rpn2 served as controls for a transmembrane and soluble protein, respectively. (D) Microsomes from HeLa cells expressing Rep8 with a C-terminal GFP tag were treated with proteinase K and Triton X-100 as indicated, before they were analyzed by SDS-PAGE and blotting. The C-terminus of Rep8 was detected by an antibody to the GFP-tag. The luminal ER protein, ERp57, served as a control.

(A) The indicated rat tissues and cell extracts were analyzed by SDS-PAGE and blotting using antibodies specific for Rep8 (upper panel) and tubulin (lower panel). Rep8 was expressed almost exclusively in testes, ovaries and in the cell types used in this study. (B) qRT-PCR for Rep8 expression during mouse testis development. Bars indicate mean expression relative to β-actin, normalized to the maximum expression level for that gene during the developmental time course. Error bars indicate standard errors. (C) qRT-PCR for Rep8 expression in adult Tex19.1^−/− testes. Mean expression levels relative to β-actin were normalized to control adult testes for each gene. Error bars indicate standard errors. (D) In situ hybridization of Rep8 to adult mouse testes. Bound sense or anti-sense Rep8 probes were visualized with dark blue/purple precipitate. Sections were counterstained with nuclear fast red. Scale bar 100 µm. (E) Higher magnification images of Rep8 in situ hybridization to adult testes. The approximate seminiferous epithelial stage is indicated by roman numerals, and examples of mitotic spermatogonia (sg), meiotic spermatocytes (sc), round spermatids (rs) and elongated spermatids (es) are annotated. Scale bar 20 µm. (F) In situ hybridization of Rep8 to adult mouse ovary. Scale bar 100 µm. Granulosa cells (gr), oocytes (oo) and corpora lutea (cl) are indicated.

(A) HeLa cells expressing in vivo biotinylated Hrd1 were precipitated using streptavidin Sepharose beads, or as a control, IgG Sepharose beads, and analyzed by SDS-PAGE and blotting using the indicated probes. Rep8 was bound to the immobilized Hrd1. (B) Human MelJuSo cells were transfected with control siRNA or two different siRNAs specific for Rep8. After 5 days, the expression of the indicated proteins was analyzed by SDS-PAGE and blotting. Actin served as a loading control. (C) Pulse-chase experiments performed on cells expressing YFP-tagged CD3δ. The cells were transfected with Rep8 siRNA#2 (filled square) or control siRNA (filled circle). At the indicated times during the chase period the substrate was retrieved by precipitation using antibodies specific for GFP. The precipitated material was resolved by SDS-PAGE and visualized by phosphoimaging (top panel). Knock-down of Rep8 expression caused a decrease in the degradation rate (lower panel). (D) The amount of membrane-bound p97 in MelJuSo cells transfected with control siRNA or siRNA specific for Rep8 was determined by differential centrifugation and quantitative blotting. The error bars show the standard deviation. The difference was significant at the p<0.01 level (**) (t-test, n = 4).