Western blot and RhoA pulldown assays

RhoA-GTP pulldown activation assay (Cytoskeleton, #BK036) was carried out according to protocol. For western blots, protein extraction was performed using RIPA buffer (Sigma) supplemented with protease/phosphatase inhibitors. Cell lysates were incubated at 37°C for 1 hour in 2xSDS buffer prior to loading on SDS-PAGE. Commercial antibodies were used in accordance to manufacturer’s guidelines: anti-LGR5 (Abcam, ab75732), anti-GPR56 (Abnova, H00009289-B01P), anti-MDR1 (Abcam, ab170904 or Cell Signaling, 13342S), anti-MRP1 (Novus, NB400-156 or Cell Signaling, 72202S), anti-ABCG2 (Cell Signaling, 42078S), anti-myc (Cell Signaling, 2276S), and anti-β-actin (Cell Signaling, 4970). HRP-labeled secondary antibodies were utilized for detection with the standard ECL protocol. Quantification was performed using ImageJ.