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. 2013 Jul;33(14):2773–2786. doi: 10.1128/MCB.00175-13

Fig 2.

Fig 2

Contribution of Net1 isoforms to LPA-stimulated RhoA activation and peripheral myosin light-chain phosphorylation. (A) MDA-MB-231 cells were transfected with control-, Net1 dual isoform-, or RhoA-specific siRNAs, starved, and then stimulated with LPA for 5 min. RhoA activation was tested by G-LISA assay. Shown is the average from three independent experiments. Errors are SEM. **, P < 0.01; ***, P < 0.001. (B) Representative Western blot of siRNA-transfected cells. (C) Immunofluorescence analysis of myosin light-chain phosphorylation 2 on serine 19 (pMLC2) in siRNA-transfected cells. Cells were starved and then stimulated with LPA for 5 min. Shown are representative maximum-intensity z-plane images. Green, pMLC2; blue, DNA. Bar, 10 μm. (D) Quantification of pMLC2 staining in the cell periphery in siRNA-transfected cells. Shown is the average from three independent experiments. Errors are SEM. *, P < 0.05; **, P < 0.01. (E) Rescue of pMLC2 in Net/Net1A siRNA-transfected cells. Shown are representative maximum-intensity z-plane images. Green, pMLC2; blue, DNA; red, transfected protein. Bar, 21 μm. (F) Quantification of pMLC2 rescue in siRNA-transfected cells. Shown is the average from three independent experiments. Errors are SEM. **, P < 0.01.