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. 2018 Apr 25;293(23):9064–9077. doi: 10.1074/jbc.RA117.001123

Figure 9.

Figure 9.

Net1-PH/RhoA heterodimer formation analysis. A, NMR data show the protein complex (200 μl at a concentration of 1. 8 mg/ml, 104.8 μm) in 20 mm Tris, 200 mm NaCl, 10% glycerol, 1 mm TCEP, pH 7.5, mixed with 20 μl of D2O and placed in a 3-mm NMR tube. B, Superdex 75 10/300 gel-filtration profile of Net1-PH (orange), RhoA (green), and Net1-PH preincubated with RhoA at a ratio of 1:1 (pink) or 2:1 (blue). The eluted fractions (1–7) are indicated with red numbers. The peak observed at 17 ml corresponds to the GDP present in the RhoA buffer. C, TGX Stain-free SDS-polyacrylamide gel of the elution fractions from the Net1-PH/RhoA gel-filtration run (ratio, 2:1). Lane 1, Net1-PH, fraction 6; lane 2, RhoA, fraction 4; lane 3, molecular mass markers, 10, 15, 20, 25, 37, 50, 75, 100, 150, and 250 kDa; lanes 4–10, elution fractions 1–7 from Net1-PH/RhoA (ratio, 2:1; blue trace in A). mAU, milli-absorbance units.