Figure S5.
IST1-immunoprecipitation and proteomics. (A) moDCs were cultured either in the presence or absence of serum followed by IST1 immunoprecipitation. Proteins were identified by mass spectrometry. Data is obtained using four donors. A.I: Flowchart of the IST1 IP. A.II: Western blot of eluted IP sample probed for IST1. A.III: Venn diagram showing proteins consistently enriched ≥1.3-fold in comparison with the sample with beads only (no antibody). The blue circle represents proteins upregulated in the samples cultured with serum and the yellow circle represents proteins upregulated in the samples cultured without serum. A.IV: Protein network showing proteins ≥1.3 times enriched in the serum-free condition over the serum-containing condition. Using the STRING tool, set at a confidence level of 0.9, proteins associated with cell adhesion were selected. Size of the dots corresponds with enrichment of the protein in the absence of serum. (B) Western blot of the pulldown confirming immunoprecipitation of the target IST1. Interaction with integrin β2 could not be detected by Western blot (expected band missing). We could not exclude that the IST1 antibody binds to IgG heavy chain, as IST1 runs at nearly the same height as the heavy chain antibody (and protein A/G). However, we confirmed the presence of IST1 with mass spec in A using the exact same procedures. (C) Confocal micrographs of moDCs immunolabeled for integrin αVβ5 (cyan in merger), IST1 (magenta) and phalloidin (yellow). For this integrin, we only observed colocalization with the ESCRT structures in one out of four tested donors. Note that integrin αVβ5 also locates to podosomes (arrow). (D) TIRF microscopy of moDCs co-expressing mCherry-labeled CHMP4 (magenta) with YFP-tagged integrin β2 (green) or only YFP (control). Graphs show the average size number of CHMP4-positive structures for cells with detectable expression of both constructs for three different donors. NS: not significant (paired two-sided t test; data distribution was assumed to be normal, but this was not formally tested). Scale bars: 10 μm. Source data are available for this figure: SourceData FS5.

IST1-immunoprecipitation and proteomics. (A) moDCs were cultured either in the presence or absence of serum followed by IST1 immunoprecipitation. Proteins were identified by mass spectrometry. Data is obtained using four donors. A.I: Flowchart of the IST1 IP. A.II: Western blot of eluted IP sample probed for IST1. A.III: Venn diagram showing proteins consistently enriched ≥1.3-fold in comparison with the sample with beads only (no antibody). The blue circle represents proteins upregulated in the samples cultured with serum and the yellow circle represents proteins upregulated in the samples cultured without serum. A.IV: Protein network showing proteins ≥1.3 times enriched in the serum-free condition over the serum-containing condition. Using the STRING tool, set at a confidence level of 0.9, proteins associated with cell adhesion were selected. Size of the dots corresponds with enrichment of the protein in the absence of serum. (B) Western blot of the pulldown confirming immunoprecipitation of the target IST1. Interaction with integrin β2 could not be detected by Western blot (expected band missing). We could not exclude that the IST1 antibody binds to IgG heavy chain, as IST1 runs at nearly the same height as the heavy chain antibody (and protein A/G). However, we confirmed the presence of IST1 with mass spec in A using the exact same procedures. (C) Confocal micrographs of moDCs immunolabeled for integrin αVβ5 (cyan in merger), IST1 (magenta) and phalloidin (yellow). For this integrin, we only observed colocalization with the ESCRT structures in one out of four tested donors. Note that integrin αVβ5 also locates to podosomes (arrow). (D) TIRF microscopy of moDCs co-expressing mCherry-labeled CHMP4 (magenta) with YFP-tagged integrin β2 (green) or only YFP (control). Graphs show the average size number of CHMP4-positive structures for cells with detectable expression of both constructs for three different donors. NS: not significant (paired two-sided t test; data distribution was assumed to be normal, but this was not formally tested). Scale bars: 10 μm. Source data are available for this figure: SourceData FS5.

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