Fig. 3.

Hrd1 and FAM8A1 interact through conserved cytoplasmic domains to enhance recruitment of Herp. Predicted intrinsic protein disorder (MetaDisorder, red/orange) and evolutionary conservation (ConSurf, blue/green) for Hrd1 (A) and FAM8A1 (B). Consensus sequences for Hrd1 (aa 484-528) and FAM8A1 (aa 106-140) generated by WebLogo 3.0 are shown below. Letter sizes correspond to their conservation. Asterisks indicate conserved residues selected for mutation. (C) Co-precipitation profiles of Hrd1-S variants lacking conserved cytoplasmic domain from A (1-499 and Δ480-535) expressed in HEK293_{Flp-In/Hrd1-KD} cells. Hrd1 1-617 (FL), 1-540 are included for comparison. Samples affinity purified by S-Ag from 1% LMNG lysates are shown along with inputs (20% of AP) with resulting blots probed for Hrd1 (S-tag), FAM8A1, SEL1L, Herp, Ube2j1 and GAPDH. Asterisk indicates a non-specific band. (D) DOX-induced expression of Hrd1 variants (1-617, L489A, R503R and Δ480-535) in HEK293_{Flp-In/Hrd1-KD} cells. Samples affinity purified by S-Ag from 1% LMNG lysates are shown along with inputs (20% of AP) with resulting blots probed for Hrd1 (S-tag), FAM8A1, SEL1L, Herp, Ube2j1, OS-9 and tubulin. (E) S-tagged FAM8A1 variants (FL, Δ107-139, W120A/W122A and W131A were transiently co-expressed along with Hrd1–Myc (FL) in HEK293_{Flp-In/Hrd1-KD} cells. Following isolation by S-Ag from cells 1% LMNG lysate, resulting blots of input (20% of AP) and AP samples were probed for Hrd1 (Myc), FAM8A1 (S-tag), Herp and tubulin. (F) Co-expression of S-DFP or S-DFP-FAM8A1_100-140 fusion protein in HEK293_Hrd1-KD cells together with Hrd1–Myc FL, TM and CYTO. Following isolation by S-Ag, the resulting blots of LMNG lysates (20% of AP) and AP samples were probed for Hrd1 (anti-Myc), FAM8A1 (anti-S-tag), Herp and GAPDH.