Figure 7.
MAP1B protects Tks5 from autophagic degradation. (A) Immunoblotting of Tks5 and cortactin in MAP1B KD and KO MDA-MB-231 cells. (B) Immunoblotting of Tks5 in MAP1S siRNA-based KD MDA-MB-231 cells. (C) RT-qPCR analysis of Tks5 mRNA levels in MAP1B KO and stable KD MDA-MB-231 cells. The values were normalized to GAPDH mRNA levels. n = 8 from four independent experiments. (D) Immunoblotting of Tks5 in proteasome- and lysosome-dependent degradation inhibitors-treated MAP1B KO MDA-MB-231 cells. Left: A representative result. Right: Quantification of Tks5 protein levels. n = 5 experiments. (E and F) Intracellular localization of GFP-Tks5 in autophagy inhibitor-treated MDA/GFP-Tks5 cells. Left: Representative immunofluorescence images of LAMP1 (E) or LC3 (F) staining in MDA/GFP-Tks5 cells treated with bafilomycin A1 or wortmannin. The arrowheads indicate the regions of colocalization between GFP-Tks5 and LAMP1 (E) or LC3 (F). Right: Quantification of the colocalization between GFP-Tks5 and LAMP1 (E) or LC3 (F). LAMP1 (E), n = 18 cells from three independent experiments; LC3 (F), n = 36 cells from three independent experiments. (G) Intracellular localization of GFP-Tks5 in MAP1B siRNA-transfected MDA/GFP-Tks5 cells. Left: Representative immunofluorescence images. The arrowheads indicate the regions of colocalization between GFP-Tks5 and LAMP1. Right: Quantification of the colocalization between GFP-Tks5 and LAMP1. Mock, n = 18 cells; MAP1B siRNA, n = 12 cells from three independent experiments. (H) Immunoblotting of Tks5 in MDA-MB-231 cells cotransfected with MAP1B and autophagy-related factor siRNAs. (C and D) The quantification values indicate the mean ± SEM. (E–G) Data are presented as boxes containing the first and third quartiles. The whiskers indicate the maxima and minima after outlier removal. (C–F) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. (G) The P value was determined using an unpaired two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. (E–G) Scale bars, 10 μm. Source data are available for this figure: SourceData F7.

MAP1B protects Tks5 from autophagic degradation. (A) Immunoblotting of Tks5 and cortactin in MAP1B KD and KO MDA-MB-231 cells. (B) Immunoblotting of Tks5 in MAP1S siRNA-based KD MDA-MB-231 cells. (C) RT-qPCR analysis of Tks5 mRNA levels in MAP1B KO and stable KD MDA-MB-231 cells. The values were normalized to GAPDH mRNA levels. n = 8 from four independent experiments. (D) Immunoblotting of Tks5 in proteasome- and lysosome-dependent degradation inhibitors-treated MAP1B KO MDA-MB-231 cells. Left: A representative result. Right: Quantification of Tks5 protein levels. n = 5 experiments. (E and F) Intracellular localization of GFP-Tks5 in autophagy inhibitor-treated MDA/GFP-Tks5 cells. Left: Representative immunofluorescence images of LAMP1 (E) or LC3 (F) staining in MDA/GFP-Tks5 cells treated with bafilomycin A1 or wortmannin. The arrowheads indicate the regions of colocalization between GFP-Tks5 and LAMP1 (E) or LC3 (F). Right: Quantification of the colocalization between GFP-Tks5 and LAMP1 (E) or LC3 (F). LAMP1 (E), n = 18 cells from three independent experiments; LC3 (F), n = 36 cells from three independent experiments. (G) Intracellular localization of GFP-Tks5 in MAP1B siRNA-transfected MDA/GFP-Tks5 cells. Left: Representative immunofluorescence images. The arrowheads indicate the regions of colocalization between GFP-Tks5 and LAMP1. Right: Quantification of the colocalization between GFP-Tks5 and LAMP1. Mock, n = 18 cells; MAP1B siRNA, n = 12 cells from three independent experiments. (H) Immunoblotting of Tks5 in MDA-MB-231 cells cotransfected with MAP1B and autophagy-related factor siRNAs. (C and D) The quantification values indicate the mean ± SEM. (E–G) Data are presented as boxes containing the first and third quartiles. The whiskers indicate the maxima and minima after outlier removal. (C–F) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. (G) The P value was determined using an unpaired two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. (E–G) Scale bars, 10 μm. Source data are available for this figure: SourceData F7.

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