Fig. 3: Rab35 effector localization and requirement for sprouting.

a Table listing each effector, respective function, and citations. b ACAP2, OCRL, MICAL-L1 and MICAL-1 knockdown (KD) validation by western blotting (ACAP2 average 70.5% KD relative to control, n = 3; OCRL average 72.9% KD relative to control, n = 3; MICAL-L1 average 61.5% KD relative to control, n = 3; MICAL-1 average 69.3% KD relative to control, n = 4). c–e Graphs of indicated sprout parameters between groups. n=number of sprouts. Error bars represent standard deviation, middle bars are the mean. f Representative images of sprout morphology between indicated siRNA (si) KD groups. Dashed lines outline microbeads. g Representative images of siRNA-mediated KD of each effector. White arrowhead denotes abnormal localization of β1-integrin. Red arrowheads denote abnormal podocalyxin (Podxl) localization. Yellow arrowheads denote abnormal actin accumulations. White dotted lines mark sprout exterior. In all images L denotes lumen. NS = non-significant. Statistical significance was assessed with an unpaired t-test or a 1-way ANOVA followed by a Dunnett multiple comparisons test. n = number of sprouts. Insets are areas of higher magnification. All experiments were done using human umbilical vein endothelial cells in triplicate.