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Fig. 2 | Molecular Cancer

Fig. 2

From: Pals1 prevents Rac1-dependent colorectal cancer cell metastasis by inhibiting Arf6

Fig. 2

Pals1 inhibits Arf6 to control Rac1-dependent cell migration and invasion. a Western blot and quantification of pulldown assays for active Cdc42 and Rac1 using the protein binding domain (PBD) of p21 activated kinase 1 protein (PAK1) (N = 4). GST-PBD was stained with Coomassie brilliant blue (CBB). Active Rac1 was quantified by normalizing against α-Actinin 4. b Quantification of wound healing assays of HCT116 and HCT116ΔPals1 with the Rac1 inhibitor EHT1864 (5 μM) and DMSO as control (N = 7). c Western blot and CBB stained gel of a pulldown experiment using recombinant constitutively active (Q67L, CA) and dominant negative (T44N, DN) Arf6 fused to GST and wild type HCT116 cell lysates. GST alone served as control. d Western blot and quantification of Arf6 expression in HCT116 and HCT116ΔPals1 cell lines (N = 4). e Western blot and quantification of the pulldown assay for active Arf6 using the effector protein Golgi-localized γ-adaptin ear-containing, Arf-binding protein 3 (GGA3) (N = 3). GST-GGA3 is visualized by CBB staining. Active Arf6 was normalized against β—Actin. f Immunostainings of migrating HCT116 and HCT116ΔPals1 cells for Arf6 (green), Rac1 (red) and Phalloidin (magenta). Arrows indicate the tips of lamellipodia. g Quantification of wound healing assays with HCT116 and HCT116ΔPals1 incubated with the Arf6 inhibitor NAV2729 (10 μM) or DMSO as control (N = 7). h Representative images and quantification of transwell matrigel invasion assays of HCT116 and HCT116ΔPals1 cells with the Arf6 inhibitor NAV2729 (10 μM), the Rac1 inhibitor EHT1864 (5 μM) or with DMSO as control (N = 4). Scale bars are 20 μm

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