![Protein purity and evaluation of the regions necessary for IQGAP1’s plus-end activity. (A) Schematic of IQGAP1 constructs and predicted molecular weights. (B) SDS-PAGE gels of IQGAP1 proteins used in this work. Incomplete cleavage of the SUMO tag results in a ∼12 kDa shift. (C) Kymographs and traces of elongating actin filaments in the presence of all IQGAP1 proteins listed in A, generated from TIRF images. Red lines in traces indicate pauses in filament elongation. +, IQGAP1 protein pauses filament elongation. −, IQGAP1 protein does not pause filament elongation. Scale for kymographs and traces: length, 3 µm; time, 100 s. (D) Duration of individual pauses measured to generate plots in E and F (n [duration] = 75–399 derived from n [filaments] = 51–324 as noted, from at least three technical replicates). Error bars, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with control (0 nM IQGAP1); (b) P ≤ 0.05 compared with 75 nM FL-IQGAP1 reactions; ns, P ≥ 0.05 to control. (E and F) Frequency distribution plots displaying the duration of IQGAP1-mediated pauses in filament elongation for all IQGAP1 conditions tested. Values for the following conditions are repeated from Fig. 2 F for comparative purposes: No IQGAP1(actin alone), IQGAP1, and FL-IQGAP1(BAD). Source data are available for this figure: SourceData FS1.](https://cdn.rupress.org/rup/content_public/journal/jcb/223/9/10.1083_jcb.202305065/2/jcb_202305065_figs1.png?Expires=1773660116&Signature=1zA3paJPUvgYKxG7atSfJ-4iDv1t3PH7PKle7FJgVnvM~GFOQNEX2swxwTeDPFvcQB~vzCoIkg2lI0Fcj2nLs2DmJrhRA2kBMgcm7q9yU5MXAeLmz6Hozw9PCP3HTswsPqW8AcjWps8KMRMTlK3Q1Oni7VGerb25wz9g6K~H~IFTOt-W8udn-Xf9f7spiRBrVuDk3Cz0Pv6y6B4kNvNPayw2sBExD2jQ~onML0I9s7bEhVPcFRIN0imI0IgGieC77jAGpQGywvwOZXB4AIWjT3Sql-aD6EbHMOv-22fUf~Iz1-sKyCkrh1OHqOaCeua~ZuL2Rty7WqANbc4e30ZYIA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Protein purity and evaluation of the regions necessary for IQGAP1’s plus-end activity. (A) Schematic of IQGAP1 constructs and predicted molecular weights. (B) SDS-PAGE gels of IQGAP1 proteins used in this work. Incomplete cleavage of the SUMO tag results in a ∼12 kDa shift. (C) Kymographs and traces of elongating actin filaments in the presence of all IQGAP1 proteins listed in A, generated from TIRF images. Red lines in traces indicate pauses in filament elongation. +, IQGAP1 protein pauses filament elongation. −, IQGAP1 protein does not pause filament elongation. Scale for kymographs and traces: length, 3 µm; time, 100 s. (D) Duration of individual pauses measured to generate plots in E and F (n [duration] = 75–399 derived from n [filaments] = 51–324 as noted, from at least three technical replicates). Error bars, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with control (0 nM IQGAP1); (b) P ≤ 0.05 compared with 75 nM FL-IQGAP1 reactions; ns, P ≥ 0.05 to control. (E and F) Frequency distribution plots displaying the duration of IQGAP1-mediated pauses in filament elongation for all IQGAP1 conditions tested. Values for the following conditions are repeated from Fig. 2 F for comparative purposes: No IQGAP1(actin alone), IQGAP1, and FL-IQGAP1(BAD). Source data are available for this figure: SourceData FS1.
