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. 2020 Jul 2;9:e57306. doi: 10.7554/eLife.57306

Figure 3. Functional associations of E3 and HCIPs.

(A) Heat map depicting established ERAD components found as HCIPs with the panel of ER-resident E3s, with colours of individual interactors corresponding to their calculated p-values. (B) Transcriptional analysis of parental Flp-In293 cells determined by NanoString. Data depict fold change of E3 transcripts measured from tunicamycin-treated (Tm, 500 ng/mL, 8 hr) and SubAB-treated (SubAB, 10 ng/mL, 8 hr) cells when compared to untreated. Mean and S.E.M. are shown from three biological repeats (n = 3). *p<0.05, **p<0.01, ***p<0.001. Detailed statistical analysis can be found in Supplementary file 1, Table 12. Hrd1 is highlighted in orange for reference. (C) Absolute number of spectral counts detected for VCP/p97 for each ER-resident E3 determined by SINQ analysis. The dotted red line shows the median spectral counts for reference (D) Heat map representing the association between proteins involved in lipid regulation (synthesis; metabolism and transport) and E3 baits. Colours associated with individual interactors correspond to their calculated p-values.

Figure 3—source data 1. Nanostring counts obtained for E3 transcripts in response to ER stressors used to generate Figure 3B.

Figure 3.

Figure 3—figure supplement 1. Validation of ER-resident E3 interactions.

Figure 3—figure supplement 1.

(A) Induction of ER stress as determined by splicing of XBP1 in Flp-In293 cells knocked down for individual ER-resident E3s by siRNAs. Splicing was validated by treatment of siNTC (non-targeting control)-transfected cells with DTT (5 mM, 2 hr). (B) Diagram representing shared HCIPs of RNF185 and RNF170. (C) Validation of RNF185 HCIPs. Transient expression of S-tagged HCIPs in Flp-In293 cells stably expressing FH-RNF185. Complexes were affinity purified from 1% LMNG-solubilised lysates by S-protein agarose, separated by SDS-PAGE and the resulting western blots probed for RNF185 (anti-HA) and the HCIP (anti-S-tag). Because of weaker expression, TMEM259-S western blots are also presented in a longer exposure. Input (IN, 20%) and affinity purified (AP) material are shown.