Fig 1: ACTN1 expression suppresses SW480 cell invasion. (A) SW480 cells expressing GFP or ACTN1-GFP (shown in green) were stained for PAX (blue) and ZYX (red) proteins and imaged as described for Fig 4A. Boxed regions in the leftmost column are enlarged and shown to the right. The arrows and arrowheads indicate PAX-positive and PAX/ZYX-double-positive plaques, respectively. Scale bar = 20 μm. (B–C) Numbers of PAX and ZYX plaques per cell were counted in GFP- or ACTN1-GFP-expressing cells. At least 66 cells were analyzed per group. Error bars refer to standard deviations between cells. n.s., not significant, *P < 0.05. (D) Ratio of ZYX-positive to PAX-positive plaques. Error bars refer to standard deviations between cells. **P < 0.01. (E) SW480 cells were transiently transfected with GFP or ACTN1-GFP, and protein expression was analyzed by western blotting using the antibodies indicated to the left of each blot. Tfx indicates transfection. The asterisk indicates a non-specific crossreacting band. (F) The invasion assay was performed using GFP- or ACTN1-GFP-expressing SW480 cells as described in the legend to Fig 6B. Invaded cells were fixed and imaged by GFP fluorescence. Scale bar = 100 μm. (G) The GFP-positive cells shown in (F) were quantified as described in the legend to Fig 6C. **P < 0.01.
Fig 2: ZYX recruitment to focal complexes in ACTN1- or ACTN4-expressing DLD-1 cells. (A) GFP, ACTN1-GFP, and ACTN4-GFP (shown in green) were expressed in DLD-1 cells, which were stained for PAX (blue) and ZYX (red) proteins and imaged under a confocal fluorescence microscope. Boxed regions in the leftmost column indicate the peripheral lamellae used for PAX and ZYX plaque quantification. A series of enlarged images of the boxed regions are shown to the right. The arrows and arrowheads indicate PAX-positive and PAX/ZYX-double-positive plaques, respectively. Scale bar = 30 µm. (B–C) Numbers of PAX and ZYX plaques per unit area of peripheral lamellae were counted in GFP-, ACTN1-, or ACTN4-expressing cells. Eight to sixteen cells were analyzed per group. Error bars refer to standard deviations between cells. n.s., not significant, *P < 0.05, **P < 0.01. (D) Ratio of ZYX-positive to PAX-positive plaques, i.e., total plaque adhesions. Error bars refer to standard deviations between cells. n.s., not significant, **P < 0.01. (E) PAX plaque size in GFP-, ACTN1- or ACTN4-expressing cells was measured in at least 171 adhesions from 8–11 cells per group. Error bars refer to standard deviations between cells. n.s., not significant, *P < 0.05, **P < 0.01.
Fig 3: Representative immunofluorescence analysis of the intracellular localization of (top) LPP and (bottom) zyxin in cultured vascular SMC isolated from the aorta of 3-month old C57BL/6 wildtype mice. The vascular SMC (passage 3) were seeded directly on microscopic slides, fixed with p-formaldehyde, and stained for LPP or zyxin with a corresponding anti-LPP (HPA017342) or anti-zyxin (HPA004835) primary antibody at a dilution of 1:75 together with a primary anti-α-smooth muscle actin (α-SMA, F3777, all Sigma-Aldrich) at a dilution of 1:200. Images were recorded using a Leica TCS SP8 laser scanning confocal microscope. Both proteins (red fluorescence) mainly localize to the interface between FA and the (tips of the) cortical actin cytoskeleton, which is chiefly organized in stress fibers indicative of a mainly quiescent contractile phenotype of the cultured vascular SMC. The nuclei were counterstained with DAPI (blue fluorescence). The size marker corresponds to 50μm.
Fig 4: ZYX binding to ACTN1 and ACTN4. (A) Pull-down assay showing that a putative ACTN-binding site, GST-ZYX-N (aa 1–51), binds specifically to His-ACTN1, but not to His-ACTN4. Bound His-ACTN1 or His-ACTN4 was analyzed by western blotting using anti-His-tag antibody. Equal amounts of input proteins were confirmed by SDS-PAGE followed by Coomassie blue staining (CBB). (B) FLAG-ZYX-full (aa 1–572) or FLAG-ZYX-C (aa 52–572) bound to anti-FLAG M2 beads were mixed either with His-ACTN1 or His-ACTN4 proteins, or without ACTN proteins (denoted by “-”). Bound His-ACTN1 or His-ACTN4 was analyzed by western blotting using anti-pan-actinin antibody. Note that the faint bands detected in lanes 1 and 4 of the pull-down blot represent non-specific background signals of pan-actinin antibody. Equal amounts of ZYX proteins were confirmed by western blotting using anti-FLAG antibody.
Fig 5: Comparison of the exon-intron structure of the primary transcripts (hnRNA) of the murine (top) Lpp and (bottom) Zyx gene and their corresponding protein products (mLPP, mZYX). The color coding for the functional elements in both proteins corresponds to that of Figure 2.
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