Fig 2: Rab40b regulates subcellular localization of EPLIN-β. (A) Control or FLAG-Rab40b-4A–expressing cells were transfected with either GFP-EPLIN-α (green) or GFP-EPLIN-β (green). Cells were then fixed and stained with phalloidin–Alexa Fluor 594 (red). Line indicates the area analyzed by line scan and shown in B. (B and C) EPLIN and actin distribution in lamellipodia was analyzed by line scan. The location of line-scan analysis is shown in A. Panel C shows the analysis of the distance between actin front at the leading edge and EPLIN front. The data shown are the means and SEM derived from five different cells. (D) Representative images of immunohistological stains of EPLIN from tumors grown from each cell line. AU, arbitrary units.

Fig 3: Rab40b–Cullin5 regulates EPLIN stability and localization. (A) Abbreviated list of proteins identified by IP of FLAG-Rab40b and FLAG-Rab40b-4A cells, followed by mass spectroscopy analysis. (B) Rab40b and EPLIN interaction analysis. Top panels: Cell lysates of FLAG-Rab40b and FLAG-Rab40b-4A cells in the presence or absence of GTPγS were incubated with either IgG or an anti-FLAG antibody and immunoprecipitated with Protein G beads. Immunoprecipitates were analyzed by WB with anti-FLAG and anti-EPLIN antibodies. Bottom left: Quantification of EPLIN and FLAG-Rab40b binding. Data are the means and SEM derived from three independent experiments. In all cases, signal was normalized to the EPLIN signal in lysate. Bottom right: MDA-MB-231 control cell lysates were incubated with glutathione beads coated with either GST or GST-Rab40b, in the presence of GDPβS or GTPγS. Bound EPLIN was eluted and analyzed by WB. (C) WBs of endogenous EPLIN in control, FLAG-Rab40b, and FLAG-Rab40b-4A cells. Quantification below are the means and SEM derived from three different experiments and normalized against tubulin levels. (D) WBs of endogenous EPLIN in control and Rab40-3KO cells. Two different 3KO lines were used in these experiments. Quantification below are the means and SEM derived from three different experiments and normalized against tubulin levels. (E) WB images of 293t cells transfected with empty vector control (CNT), FLAG-EPLIN-α, or FLAG-EPLIN-β for 24 h; treated with 10 µm MG132 overnight; harvested and immunoprecipitated for FLAG; and then blotted for either ubiquitin (top) or FLAG (bottom). (F) WB images of 293t cells treated with siRNAs for nontargeting control (siCNT), Rab40b, Rab40c, or both Rab40b and Rab40c; transfected with FLAG-EPLIN-α; treated with MG132 overnight; harvested and immunoprecipitated for FLAG; and then blotted for either ubiquitin (top) or FLAG (bottom). Quantification on the right shows the means and SEM derived from three different experiments. KD, knockdown.

Fig 4: GFP-Rab40b colocalizes at the actin ruffles at the leading edge of lamellipodia. (A–F) Representative images of GFP-Rab40b (A, C, and E) or GFP-Rab40b-4A (B, D, and F) expressing MDA-MB-231 cells plated on collagen-coated coverslips and then fixed and stained with phalloidin–Alexa Fluor 594 (see insets in A and B), anti-paxillin (C and D), or anti-EPLIN antibodies (E and F). Boxes mark the region of interest shown in the inset. Asterisks mark the leading edge of lamellipodia. Arrowheads mark EPLIN staining that does not colocalize with GFP-Rab40b. Arrows point to structures positive for both EPLIN and GFP-Rab40b-4A.

Fig 5: Rab40b inhibits EPLIN accumulation at the lamellipodia leading edge. (A) Schematic representation of FA and stress fiber distribution in lamellipodia. (B–G) Representative line scans from control (E), FLAG-Rab40b-4A (F), and Rab40-3KO (G) cells plated on collagen-coated coverslips and then fixed and stained with phalloidin–Alexa Fluor 594 (red) and anti-EPLIN (green) antibodies. Boxes mark the region of interest shown in inset. Line marks the region analyzed by line scan (quantifications shown in B–D). EPLIN distance from actin front: control, 1.8 µm ± 1.1; FLAG-Rab40b-4A, 0.68 µm ± 0.33 (P < 0.05); and Rab40-3KO, 0.36 µm ± 0.29 (P < 0.02). n = 5 cells for each cell line.