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. 2020 Jul 20;18(7):e3000606. doi: 10.1371/journal.pbio.3000606

Fig 1. Global identification of HSPA1A and HSPA8 clients using UBAIT in human cells.

Fig 1

(A) Schematic diagram of biotin/V5-HSP70/HSC70 UBAIT conjugation to bound targets. (B) Western blot of HSP70 and HSC70 UBAITs after streptavidin isolation from human U2OS cells treated with doxycycline (Dox) (1 ug/mL) for 3 days or untreated. (C) Schematic of experimental and statistical workflow used for the analysis of UBAIT-conjugated targets. Twelve wild-type or ΔGG samples are first analyzed by LC-MS/MS using label-free quantification, then missing values are imputed by low abundance resampling (5%). Wild-type and ΔGG values are used to calculate an enrichment ratio and also are bootstrapped to generate random enrichment ratio distribution (1,000×). The actual enrichment ratio is compared with the bootstrapped distribution to obtain p-values, which are corrected for multiple testing hypotheses using the Benjamini-Hochberg method. (D) Venn diagrams of HSC70 and HSP70 targets identified from 3 independent experiments, each with N = 12, all with K48R ubiquitin fusions. (E) Venn diagram of proteins identified as significantly enriched from all three experiments compared with the ubiquitin-only control. (F) Co-chaperones and known chaperone interactors identified from (D). All factors shown were identified at FDR 0.05. Black outline: enriched for HSC70 and HSP70 binding in all 3 sets for both chaperones; red outline: enriched for HSC70 in all 3 sets, no enrichment for HSP70; blue outline: enriched for HSC70 in all 3 sets, enriched for HSP70 in at least one set; green outline: enriched for HSP70 in all 3 sets, no enrichment for HSC70; purple outline: enriched for HSP70 in all 3 sets, enriched for HSC70 in at least one set. Fill colors show chaperones (gray), DNAJ proteins (green), nucleotide exchange factors (blue), other co-chaperones (orange or pink), and chaperonins (purple). Bio-V5, biotin-V5 tag; DNAJ, heat shock protein 40; FDR, false discovery rate; HSC, heat shock cognate; HSP, heat shock protein; IP, immunoprecipitation; LC-MS/MS, liquid chromatography–mass spectrometry; Ub, ubiquitin; UBAIT, ubiquitin-activated interaction trap; WT, wild-type.