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Fig. 8 | BMC Biology

Fig. 8

From: An RNAi screen of Rho signalling networks identifies RhoH as a regulator of Rac1 in prostate cancer cell migration

Fig. 8

RhoH alters the distribution of active Rac1. a RhoH does not affect total Rac1 activity. PC3 cells transfected with the indicated siRNAs were analysed for Rac1 activity using GST-PBD and pulldown assays. Blot is representative of three independent experiments. The ratio of GTP-Rac1/Total Rac1 (compared to siControl) is indicated under the blot panels. b RhoH does not affect RhoA or Cdc42 activity. PC3 cells transfected with the indicated siRNAs were analysed for Cdc42 (top) or RhoA (bottom) activity using GST-RBD or GST-PBD beads and pulldown assays. Blot is representative of three independent experiments. Graph shows the ratio of GTP-RhoA/Total RhoA or GTP-Cdc42/Total Cdc42 (compared to siControl); n = 3, ns non-significant, **p ≤ 0.01, Student’s t test. ce RhoH affects Rac1 activity distribution. 24 h after transfection with the indicated siRNAs, cells were transfected with DNA encoding a Rac1 biosensor including YPet PAK1-PBD and CyPet Rac1. Cells were imaged 24 h later by time-lapse microscopy on a wide-field microscope by epifluorescence. c Two example images of Rac1 activity distribution are shown for each siRNA. Scale bars 20 μm. d Graph of line scans (12 cells × 60 frames per cell) showing Rac1 activity with respect to distance from the edge of the cell and also depending on whether the cell edge was protruding or retracting. e Rac1 activity as a function of distance from cell edge and speed of membrane protrusion or retraction, n = 12 cells per condition

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