Stress fiber formation and MMP secretion analysis in Rab40b-4A and Rab40-3KO cells. (A) Control, FLAG-Rab40b, and FLAG-Rab40-4A cell lines were plated on collagen-coated coverslips and fixed and stained with anti-cortactin (image on left) and anti-CD63 antibodies (image on right). Boxes mark the region that is displayed as higher-resolution image. Arrows point to organelles positive for both cortactin and CD63. Quantification on the left are the means and SEM. (B) WB images of Tks5 and p130Cas from control and FLAG-Rab40b-4A cells. Numbers represent densitometry analysis from one biological replicate relative to tubulin and standardized to control levels. (C) The knockdown efficiency for Rab40b siRNA and Rab40c siRNA as determined by RT-qPCR. (D) Control, Rab40-3KO-1, and Rab40-3KO-2 cell lines were plated on collagen-coated coverslips and then fixed and stained using phalloidin–Alexa Fluor 594. Boxes mark the region that is displayed as higher-resolution image. Arrows point to stress fibers. Quantification on the right shows the means and SEM derived from three independent analyses. A total of 150 cells were analyzed for each condition. (E) Gelatin zymography analysis of MMP2 and MMP9 secretion from control, FLAG-Rab40b, and FLAG-Rab40b-4A cells grown in serum-free media for 24 h. FBS (serum) contains MMP2/9 and was used as a positive control. The data shown are the means and SEM derived from three independent experiments. The data were normalized against serum (S) levels of MMP2 and MMP9. DKD, double knockdown.