Figure 3. KBP inhibits KIF15 and KIF18A... | Rockefeller University Press

Figure 3.

$Figure 3. KBP inhibits KIF15 and KIF18A motor function in vitro. (A) Gliding filament assay with KIF18A-N480-GFP-His6 and GST-KBP. Left: Representative fields showing the first frame (red) and last frame (green) of MT filaments in the presence or absence of GST-KBP. Scale bars, 20 µm. Full movies (Videos 1 and 2) are available in the online supplemental material. Right: Quantification of percent inhibition of MT gliding for KIF18A-N480-GFP-His6 alone (n = 95) or with 100 nM (n = 147) or 250 nM (n = 123) GST-KBP present in the flow cell. N = total number of MTs measured from triplicate independent experiments. (B) Gliding filament assay with His6-KIF15-N700 and GST-KBP. Left: Representative fields showing the first frame (red) and last frame (green) of MT filaments in the presence or absence of GST-KBP. Full movies (Videos 3 and 4) are available in the online supplemental material. Right: Quantification of MT gliding velocity for His6-KIF15-N700 alone (n = 76) or with 250 nM (n = 125) or 500 nM GST-KBP (n = 92) present in the flow cell. N = total number of MTs measured from triplicate independent experiments. Box and whisker plots denote the 10th, 25th, 75th, and 90th percentiles. ****, adjusted P < 0.0001 with 95% confidence interval by one-way ANOVA with Tukey’s multiple comparisons test as compared with control gliding reactions. (C) Western blot showing coIP of purified 50 nM KIF18A-N480-GFP-His6 (top) or 50 nM His6-KIF15-N420 (bottom) with 250 nM GST-KBP probed with antibodies targeting 6xHis (α His) and KBP (α KBP). (D and E) Results of co-pelleting assays for KIF18A-N480-GFP-His6 (D) and His6-KIF15-N420 (E) in the presence and absence of GST-KBP and/or MTs. P, pellet; S, supernatant. Representative Coomassie gels are shown along with quantification of percentage of kinesin motor present in co-pelleting fractions. Data were obtained from three independent experiments. ns, not significant; ****, adjusted P < 0.0001; *, adjusted P = 0.031 with 95% confidence interval by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM.$

KBP inhibits KIF15 and KIF18A motor function in vitro. (A) Gliding filament assay with KIF18A-N480-GFP-His₆ and GST-KBP. Left: Representative fields showing the first frame (red) and last frame (green) of MT filaments in the presence or absence of GST-KBP. Scale bars, 20 µm. Full movies (Videos 1 and 2) are available in the online supplemental material. Right: Quantification of percent inhibition of MT gliding for KIF18A-N480-GFP-His₆ alone (n = 95) or with 100 nM (n = 147) or 250 nM (n = 123) GST-KBP present in the flow cell. N = total number of MTs measured from triplicate independent experiments. (B) Gliding filament assay with His₆-KIF15-N700 and GST-KBP. Left: Representative fields showing the first frame (red) and last frame (green) of MT filaments in the presence or absence of GST-KBP. Full movies (Videos 3 and 4) are available in the online supplemental material. Right: Quantification of MT gliding velocity for His₆-KIF15-N700 alone (n = 76) or with 250 nM (n = 125) or 500 nM GST-KBP (n = 92) present in the flow cell. N = total number of MTs measured from triplicate independent experiments. Box and whisker plots denote the 10th, 25th, 75th, and 90th percentiles. ****, adjusted P < 0.0001 with 95% confidence interval by one-way ANOVA with Tukey’s multiple comparisons test as compared with control gliding reactions. (C) Western blot showing coIP of purified 50 nM KIF18A-N480-GFP-His₆ (top) or 50 nM His₆-KIF15-N420 (bottom) with 250 nM GST-KBP probed with antibodies targeting 6xHis (α His) and KBP (α KBP). (D and E) Results of co-pelleting assays for KIF18A-N480-GFP-His₆ (D) and His₆-KIF15-N420 (E) in the presence and absence of GST-KBP and/or MTs. P, pellet; S, supernatant. Representative Coomassie gels are shown along with quantification of percentage of kinesin motor present in co-pelleting fractions. Data were obtained from three independent experiments. ns, not significant; ****, adjusted P < 0.0001; *, adjusted P = 0.031 with 95% confidence interval by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM.