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. 2012 Sep 3;198(5):865–880. doi: 10.1083/jcb.201204107

Figure 2.

Figure 2.

Arginine finger mutations reduce CYK4 GAP activity toward Rac1. (A) A schematic of CYK4 shows the conserved coiled coil, C1 putative membrane-binding region, and GAP domain with arginine finger residue at amino acid 385. Recombinant human ARHGAP1, CYK4, CYK4R385A, CYK4S387A, and CYK4S387D were tested against RhoA, Rac1, Cdc42, and Rab1. The amount of GAP-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. (B). HeLa cells were transfected with Myc-tagged CYK4, CYK4R385A, CYK4S387A, and CYK4S387D for 24 h, fixed, and then stained with antibodies to the Myc epitope, MKlp1, or tubulin. DNA was stained with DAPI. Bar, 10 µm. (C) HeLa cells stably expressing inducible Myc-tagged copies of CYK4, CYK4R385A, CYK4S387A, and CYK4S387D were treated for 36 h with an siRNA directed to the 3′-UTR of CYK4 to deplete the endogenous protein or a control siRNA. At the same time, the cells were treated with doxycycline to induce the Myc-tagged transgenes. MKlp1–CYK4 centralspindlin complexes were then immune precipitated using Myc antibodies. Control isolations were performed using GFP antibodies. These complexes were then used for GAP assays with RhoA and Rac1. The amount of centralspindlin-dependent GTP hydrolysis in picomoles per hour was calculated and plotted as a bar graph. Aliquots of the total cell lysate (input) and isolated complexes were Western blotted for Myc, CYK4, MKlp1, and tubulin. Arrowheads indicate endogenous CYK4, and arrows show Myc-tagged CYK4. Error bars indicate the standard deviations. IP, immunoprecipitation; WT, wild type.