Fig. 6.

Denaturing condition-resistant FIH interactome composition. (A) Following ectopic expression of the indicated proteins, HEK293 cells were lysed under native or denaturing (boiling in 1% SDS) conditions followed by anti-FLAG IP and immunoblot analysis. HD, heterodimer; M, monomer; ab, antibody. (B) Following ectopic expression of either FIH-V5 or tandem EGFP, HEK293 cells were lysed as described in (A) followed by anti-V5 IP and immunoblot analysis. Arrow heads indicate protein signals that were only detected following IP from native samples (-SDS). *, protein signals detected in IPs from both conditions (native and denatured lysates); exp, exposure. (C) Following the same sample preparation as in (B), the samples were analysed by MS. Venn diagram displaying the overlap between FIH interactors under native (-SDS) and denaturing (+SDS) conditions. (D) Rank order of the 13 proteins interacting with FIH under both conditions shown in (C), according to the relative label free quantification (LFQ) intensity following IP in the presence of SDS and normalized to FIH pull-down. Data are shown as mean ± SEM. HD, heterodimer; M, monomer; ab, antibody; exp, exposure. Data are shown as mean ± SEM from (C, D) four biological replicates or are representative for (A) three or (B) one independent experiment. (B–D) Samples were processed in parallel and only differ in the analysis (immunoblot or MS).