Figure 4.
MAP1B is associated with cortactin. (A) Immunofluorescence staining of MAP1B-LC1 and cortactin in MDA-MB-231 cells with or without digitonin permeabilization before fixation. Left: Representative immunofluorescence images. The arrowheads indicate the regions of colocalization between MAP1B-LC1 and cortactin. Right: Quantification of colocalization (Pearson’s coefficient) with or without 90° rotation of a color channel. n = 6 (− digitonin) and 7 (+ digitonin) cells from three independent experiments. (B) Coimmunoprecipitation of cortactin-GFP WT and its mutants with FLAG-MAP1B-LC1 in 293T cells. Upper panel: Immunoblots. Lower panel: Domain structures of cortactin and its mutants, and summary of the immunoprecipitation results. (C) Upper panel: Potential SH3 domain-binding motifs, PxxP or K/R-P, in MAP1B-LC1. The motifs and substituted residues are underlined and orange-colored, respectively. Lower panel: Coimmunoprecipitation of cortactin-GFP with FLAG-MAP1B-LC1 mutants. Right panel: Quantification of cortactin-GFP coprecipitated with FLAG-MAP1B-LC1 WT and mutants. n = 4 experiments. (D) Quantification of gelatin degradation at invadopodia in MDA-MB-231 cells with MAP1B depletion and re-expression of WT and P145A/P146A cortactin-less binding mutant of MAP1B. n = 4 experiments. (E) PLA between MAP1B-LC1 and cortactin using antibodies against both or either of them. Left: Representative immunofluorescence images. Right: Quantification of PLA dots. n = 30 cells from three independent experiments. (F) PLA between MAP1B-LC1 and cortactin in MDA-MB-231 cells. The arrowheads indicate PLA signals proximal to invadopodia-like actin dots. (G) PLA between MAP1B-LC1 and cortactin in latrunculin A- and nocodazole-treated MDA-MB-231 cells. Left: Representative immunofluorescence images. Right: Quantification of the PLA dots. Vehicle, n = 17 cells; latrunculin A and nocodazole, n = 12 cells from three independent experiments. (H) Coimmunoprecipitation of cortactin-GFP with FLAG-MAP1B-LC1 in latrunculin A- and nocodazole-treated 293T cells. (C and D) The values indicate the mean ± SEM. (E and G) Data are presented as boxes containing the first and third quartiles. The whiskers indicate the maxima and minima after outlier removal. (C–E and G) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. (A and E–G) Scale bars, 10 μm. Source data are available for this figure: SourceData F4.

MAP1B is associated with cortactin. (A) Immunofluorescence staining of MAP1B-LC1 and cortactin in MDA-MB-231 cells with or without digitonin permeabilization before fixation. Left: Representative immunofluorescence images. The arrowheads indicate the regions of colocalization between MAP1B-LC1 and cortactin. Right: Quantification of colocalization (Pearson’s coefficient) with or without 90° rotation of a color channel. n = 6 (− digitonin) and 7 (+ digitonin) cells from three independent experiments. (B) Coimmunoprecipitation of cortactin-GFP WT and its mutants with FLAG-MAP1B-LC1 in 293T cells. Upper panel: Immunoblots. Lower panel: Domain structures of cortactin and its mutants, and summary of the immunoprecipitation results. (C) Upper panel: Potential SH3 domain-binding motifs, PxxP or K/R-P, in MAP1B-LC1. The motifs and substituted residues are underlined and orange-colored, respectively. Lower panel: Coimmunoprecipitation of cortactin-GFP with FLAG-MAP1B-LC1 mutants. Right panel: Quantification of cortactin-GFP coprecipitated with FLAG-MAP1B-LC1 WT and mutants. n = 4 experiments. (D) Quantification of gelatin degradation at invadopodia in MDA-MB-231 cells with MAP1B depletion and re-expression of WT and P145A/P146A cortactin-less binding mutant of MAP1B. n = 4 experiments. (E) PLA between MAP1B-LC1 and cortactin using antibodies against both or either of them. Left: Representative immunofluorescence images. Right: Quantification of PLA dots. n = 30 cells from three independent experiments. (F) PLA between MAP1B-LC1 and cortactin in MDA-MB-231 cells. The arrowheads indicate PLA signals proximal to invadopodia-like actin dots. (G) PLA between MAP1B-LC1 and cortactin in latrunculin A- and nocodazole-treated MDA-MB-231 cells. Left: Representative immunofluorescence images. Right: Quantification of the PLA dots. Vehicle, n = 17 cells; latrunculin A and nocodazole, n = 12 cells from three independent experiments. (H) Coimmunoprecipitation of cortactin-GFP with FLAG-MAP1B-LC1 in latrunculin A- and nocodazole-treated 293T cells. (C and D) The values indicate the mean ± SEM. (E and G) Data are presented as boxes containing the first and third quartiles. The whiskers indicate the maxima and minima after outlier removal. (C–E and G) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. (A and E–G) Scale bars, 10 μm. Source data are available for this figure: SourceData F4.

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