Figure S4.
MAP1B-LC1 directly interacts with SH3 domains of cortactin and Tks5, and the expression levels of Tks5 in MAP1B- and autophagy genes-depleted MDA-MB-231 cells. (A) In vitro GST pull-down assays using purified His-MAP1B-LC1 and GST-cortactin SH3 domain fusion proteins. (B) In vitro GST pull-down assays using purified His-MAP1B-LC1 and GST-Tks5 SH3 domain fusion proteins. (C) Left panel: Coimmunoprecipitation of GFP-Tks5 with FLAG-MAP1B-LC1 mutants in 293T cells. Right panel: Quantification of GFP-Tks5 coprecipitated with FLAG-MAP1B-LC1 WT and mutants. n = 4 experiments. (D) Left panel: Tripartite coimmunoprecipitation of GFP-MAP1B-LC1 and cortactin-GFP with FLAG-Tks5 in 293T cells. Right panel: Quantification of GFP-MAP1B-LC1 and cortactin-GFP coprecipitated with FLAG-Tks5. n = 3 experiments. (E) Upper panel: In vitro GST pull-down assays using purified His-MAP1B-LC1, His-Tks5 aa 817–1118, and GST-Tks5 Pro-SH3 (see A and B for their regions). Lower panel: Quantification of His-MAP1B-LC1 and His-Tks5 aa 817–1118 precipitated with GST-Tks5 Pro-SH3. n = 3 experiments. (F) RT-qPCR analysis of Tks5 and GAPDH mRNA levels in MAP1B KO and stable KD MDA-MB-231 cells. n = 8 from four independent experiments. (G) RT-qPCR analysis of ULK1, ATG9A, and ATG14L mRNA levels in ULK1, ATG9A, or ATG14L KD MDA-MB-231 cells. n = 6 from three independent experiments. The values were normalized to GAPDH mRNA levels. (H) Immunoblotting of Tks5 in MAP1B KO MDA-MB-231 cells transfected with ULK1 siRNAs. (C–G) The values indicate the mean ± SEM. (C and F) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. (D, E, and G) P values were 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. Source data are available for this figure: SourceData FS4.

MAP1B-LC1 directly interacts with SH3 domains of cortactin and Tks5, and the expression levels of Tks5 in MAP1B- and autophagy genes-depleted MDA-MB-231 cells. (A) In vitro GST pull-down assays using purified His-MAP1B-LC1 and GST-cortactin SH3 domain fusion proteins. (B) In vitro GST pull-down assays using purified His-MAP1B-LC1 and GST-Tks5 SH3 domain fusion proteins. (C) Left panel: Coimmunoprecipitation of GFP-Tks5 with FLAG-MAP1B-LC1 mutants in 293T cells. Right panel: Quantification of GFP-Tks5 coprecipitated with FLAG-MAP1B-LC1 WT and mutants. n = 4 experiments. (D) Left panel: Tripartite coimmunoprecipitation of GFP-MAP1B-LC1 and cortactin-GFP with FLAG-Tks5 in 293T cells. Right panel: Quantification of GFP-MAP1B-LC1 and cortactin-GFP coprecipitated with FLAG-Tks5. n = 3 experiments. (E) Upper panel: In vitro GST pull-down assays using purified His-MAP1B-LC1, His-Tks5 aa 817–1118, and GST-Tks5 Pro-SH3 (see A and B for their regions). Lower panel: Quantification of His-MAP1B-LC1 and His-Tks5 aa 817–1118 precipitated with GST-Tks5 Pro-SH3. n = 3 experiments. (F) RT-qPCR analysis of Tks5 and GAPDH mRNA levels in MAP1B KO and stable KD MDA-MB-231 cells. n = 8 from four independent experiments. (G) RT-qPCR analysis of ULK1, ATG9A, and ATG14L mRNA levels in ULK1, ATG9A, or ATG14L KD MDA-MB-231 cells. n = 6 from three independent experiments. The values were normalized to GAPDH mRNA levels. (H) Immunoblotting of Tks5 in MAP1B KO MDA-MB-231 cells transfected with ULK1 siRNAs. (C–G) The values indicate the mean ± SEM. (C and F) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. (D, E, and G) P values were 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. Source data are available for this figure: SourceData FS4.

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