Figure S3. OSBP proximity labelling and... | Rockefeller University Press

Figure S3.

$OSBP proximity labelling and cell surface proteomics. (A) Domain organization of the OSBP-TurboID-HA construct stably expressed in MDCK cells. (B) Confocal imaging shows that incubating OSBP-TurboID expressing MDCK cells for 2 h after biotin treatment results in the enrichment of biotinylation signal at cell-cell junctions (arrowheads). Bar = 12 μm. (C) Experimental design and data analysis strategy of the TurboID experiment. (D) Biotinylation profiles of cell lysates obtained from wild-type MDCK or MDCK-OSBP-TurboID-HA cells. Membranes probed by streptavidin-HRP confirm successful protein biotinylation in MDCK cells stably expressing OSBP-TurboID-HA. Note that ORPphilin treatments affect the biotinylation profile (left panel). Elution profiles of streptavidin beads incubated with lysates obtained from wild-type MDCK or MDCK-OSBP-TurboID-HA cells. Biotinylated proteins were enriched on streptavidin beads; then beads were eluted by boiling, elution fractions were resolved on SDS-PAGE and revealed by silver staining (right panel). Elution profiles are comparable to biotinylation profiles shown in the left panel. (E) GO cellular component enrichment analyses show significant enrichment in multiple GO classes. (F) No correlation between polarity value and abundancy in the OSBP proximity proteome can be established, indicating that cargoes with both apical and basolateral distribution can be identified among the OSBP proximity partners. (G) Surface proteins identified by Caceres et al. (2019) were ranked according to their distribution on the polarized MDCK cell surface (Polarity value). Red dots represent surface proteins identified also in the OSBP proximity proteome. Based on this ranking, highly apical and highly basolateral proteins were selected and subsequently mapped to the OSBP proximity proteome as shown in Fig. 3, G and H. (H) Experimental design and data analysis strategy applied for the surface proteomics experiment presented in Fig. 4. Source data are available for this figure: SourceData FS3.$

OSBP proximity labelling and cell surface proteomics. (A) Domain organization of the OSBP-TurboID-HA construct stably expressed in MDCK cells. (B) Confocal imaging shows that incubating OSBP-TurboID expressing MDCK cells for 2 h after biotin treatment results in the enrichment of biotinylation signal at cell-cell junctions (arrowheads). Bar = 12 μm. (C) Experimental design and data analysis strategy of the TurboID experiment. (D) Biotinylation profiles of cell lysates obtained from wild-type MDCK or MDCK-OSBP-TurboID-HA cells. Membranes probed by streptavidin-HRP confirm successful protein biotinylation in MDCK cells stably expressing OSBP-TurboID-HA. Note that ORPphilin treatments affect the biotinylation profile (left panel). Elution profiles of streptavidin beads incubated with lysates obtained from wild-type MDCK or MDCK-OSBP-TurboID-HA cells. Biotinylated proteins were enriched on streptavidin beads; then beads were eluted by boiling, elution fractions were resolved on SDS-PAGE and revealed by silver staining (right panel). Elution profiles are comparable to biotinylation profiles shown in the left panel. (E) GO cellular component enrichment analyses show significant enrichment in multiple GO classes. (F) No correlation between polarity value and abundancy in the OSBP proximity proteome can be established, indicating that cargoes with both apical and basolateral distribution can be identified among the OSBP proximity partners. (G) Surface proteins identified by Caceres et al. (2019) were ranked according to their distribution on the polarized MDCK cell surface (Polarity value). Red dots represent surface proteins identified also in the OSBP proximity proteome. Based on this ranking, highly apical and highly basolateral proteins were selected and subsequently mapped to the OSBP proximity proteome as shown in Fig. 3, G and H. (H) Experimental design and data analysis strategy applied for the surface proteomics experiment presented in Fig. 4. Source data are available for this figure: SourceData FS3.