(A) HCIP interaction network wheel for FH-RNF26Y432A. Legend described in Figure 2—figure supplement 1. (B) Venn diagram of HCIPs identified by LC-MS/MS for FH-RNF26WT and FH-RNF26Y432A. ER-resident HCIPs are indicated in blue. (C) Co-precipitation of FH-RNF26WT from stable Flp-In293 cells by transiently expressed S-tagged HCIPs (TMED1, TMEM43, UBXD8 and AUP1). Cells were solubilised in 1% LMNG and protein complexes affinity purified from the resulting lysates by S-protein agarose. Western blots of affinity purified material (AP) and input lysate (IN, 20%) were probed with antibodies recognising RNF26 (HA), HCIPs (S-tag), TMEM33 and ENDOD1. (D) Velocity sedimentation of FH-RNF26 complexes from 1% LMNG-solubilised lysates on a sucrose gradient (5–30%), with individual TCA-precipitated fractions (1-13) subsequently separated by SDS-PAGE and the resulting western blots probed for the indicated proteins. Ubiquitinated forms of RNF26 are indicated by black arrowheads. Molecular weight of gel filtration standards solubilised and sedimented in equivalent buffer conditions are shown for comparison beneath the Hrd1 blot, which in turn serves to highlight a complex of a different mass. (E) siRNA-mediated knockdown of HCIPs in FH-RNF26WT Flp-In293 cells alters interaction profiles. RNF26 complexes immunoprecipitated by anti-FLAG agarose were separated by SDS-PAGE with resulting western blots probed by antibodies for RNF26 and the indicated HCIPs. Tubulin was used as a loading control. Ubiquitinated forms of RNF26 are indicated by black arrowheads. (F) Cycloheximide (CHX) chase assays (100 μg/ml; 0, 1, 2 hr) of FH-RNF26WT Flp-In293 cells (DOX, 1 µg/mL, 18 hr) knocked down for individual HCIPs by siRNAs with the resulting western blots probed for the indicated antibodies. MG132 (10 µM, 2 hr) and NMS-873 (10 µM, 2 hr) were included with NTC samples where indicated. Ubiquitinated forms of RNF26 are denoted by black arrowheads. Mature and immature forms of CD147 are indicated by black arrowheads.