Fig. 7: Phosphorylation-sensitive recruitment of Dok1 supports invadopodia formation.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a, A schematic of the interaction between ITGB1-V1 and a V2-tagged adaptor or regulator, highlighting the resulting V1/V2 (Venus) protein complex as the restored epitope for BiCAP (top) or donor for BiFC–FLIM–FRET (bottom). The Venus tag alone was used as a control for the BiCAP and BiFC–FLIM–FRET experiments. b, A representative BiCAP immunoblot after HEK293T cell transfection with either Venus or ITGB1-V1/Dok1-V2 (n = 3 biological replicates). c, Representative BiCAP immunoblots from MM231 and HEK293T cells where annexin A6 (AnxA6), VPS35 and Cofilin coimmunoprecipitate with the Dok1/ITGB1 complex (n = 3 biological replicates). d, Representative BiFC–FLIM–FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 cells transfected with RFP-tagged Cofilin mutants WT, S3A and S3E (Venus, n = 73 (WT), 72 (S3A) and 64 (S3E); BiFC, n = 92 (WT), 64 (S3A) and 64 (S3E) cells from three biological replicates; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons). Scale bars, 20 μm. e, Representative images (left) and quantification of gelatin degradation (right) by MM231 ITGB1(WT or YYFF) cells with Dox-inducible Src(E378G) expression. The MM231 cells were transfected with siRNAs against Dok1 (siDok1_1 and siDok1_2) or a NTC siRNA and treated with Dox for 24 h before being seeded on fluorescent gelatin (green) for 6 h (actin labelled with SiR-actin (white), nuclei with DAPI (blue)) (ITGB1(WT), n = 36 (NTC), 38 (siDok1_1) and 36 (siDok1_2); ITGB1(YYFF), n = 38 (NTC), 34 (siDok1_1) and 37 (siDok1_2) fields of view pooled from three biological replicates; one-way ANOVA with a Šidák correction for multiple comparisons). Scale bars, 20 μm; insets, 10 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas highlight the IQR of the control conditions. NS, not significant. ***P < 0.001.
