Fig 1: Deactivation of DIAPH1 requires loss of binding to RhoA and IQGAP1.a HeLa cell fixed and probed with an antibody recognizing RhoA, white and DAPI stained chromosomes in blue. b Preformed complexes of His6-DIAPH1-NT with GST-RhoA, GST-IQGAP1 or both prevent MBP-DIAPH1-CT binding to His6-DIAPH1-NT. Schematic depicts that DIAPH1 deactivation requires the removal of both RhoA and IQGAP1. CT = C-terminus.
Fig 2: The CLIP170-IQGAP1 interaction is dependent upon anaphase astral microtubules.a Time-lapse images of GFP-CLIP170 during cell division. b Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 (top), IQGAP1 to CLIP170 (middle) and CLIP170 to DIAPH1 (bottom) during mitotic progression. c Quantitation of PLA foci in metaphase and anaphase *p < 0.0001 using a nonparametric two-tailed Mann-Whitney t-tests for three experimental repeats, n = 50 cells examined over three independent experiments. d Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 and IQGAP1 to CLIP170 in Centrinone B-treated cells. e Quantitation of PLA foci in d. At cell poles lacking astral microtubules, DIAPH1 and IQGAP1 are in close proximity but IQGAP1 and CLIP170 are not. *p = 0.0006, **p < 0.0001 using nonparametric two-tailed Mann-Whitney t-tests for three experimental repeats, n = 50 cells examined over three independent experiments. Scale bars are 10 µm.
Fig 3: IQGAP1 preferentially binds to CLIP170 over DIAPH1.a Preassembled GST-IQGAP1-DBR and His6-DIAPH1-NT complexes were incubated with MBP-CLIP170-NT. In parallel preassembled GST-IQGAP1-DBR and MBP-CLIP170-NT complexes were incubated with His6-DIAPH1-NT. After reisolating GST-IQGAP1-DBR, the amount of co-purifying MBP-CLIP170-NT or His6-DIAPH1-NT was determined. NT = N-terminus, DBR = Diaphanous Binding Region. b Schematic representing inferred IQGAP1 interaction dynamics.
Fig 4: Cortical ?-actin is required for cytokinesis.a Dividing HeLa cells fixed and stained with antibodies recognizing ß-actin, ?-actin, DIAPH1, DIAPH3, and IQGAP1. b Quantitation of the number of multinucleated cells in the presence and absence of DIAPH1, IQGAP1, and DIAPH1-I862A that cannot nucleate actin. Bars denote the average, whiskers denote ±SD. *p = 0.0001, **p < 0.0001 to control using nonparametric two-tailed Mann-Whitney t-tests for three experimental repeats, n = 600 cells examined over three independent experiments. c ß-actin or ?-actin localization in HeLa cells treated with control siRNA or DIAPH1 siRNA in the presence and absence of exogenously expressed GFP-DIAPH1 or GFP-DIAPH1-I862A. d Frames from time-lapse phase contrast movies demonstrating that depletion of DIAPH1 caused an increase in plasma membrane blebbing during anaphase. White arrows denote, blebs, black arrows point to the cytokinetic furrow. e Quantitation of the number of plasma membrane blebs in fixed metaphase and anaphase cells. Bars denote the average, boxes represent 25–75 percentile and whiskers denote extremes. *p = 0.014, **p = 0.0007, ***p = 0.0006, ****p = 0.0005, *****p = 0.0004 difference to control using nonparametric two-tailed Mann-Whitney t-tests for three experimental repeats, n = 300 cells examined over three independent experiments. CT = C terminus; DIA1 = DIAPH1; DIA3 = DIAPH3; IQ1 = IQGAP1. f Quantitation of the rate of cell elongation from 2–8 min after anaphase onset. Bars denote the average, whiskers denote ±SD. *p = 0.029, **p = 0.005, ***p = 0.0007, ****p = 0.0003, *****p < 0.0001 to control, using nonparametric two-tailed Mann-Whitney t-tests for three experimental repeats, n = 10 cells examined in each live-cell imaging group. CT = C terminus; DIA1 = DIAPH1; DIA3 = DIAPH3. Scale bars are 10 µm.
Supplier Page from Abcam for Anti-IQGAP1 antibody [EPR5221]