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Fig. 4. Expression of N-cadherin, MRTF-A, sm MHC, nm MHC and PP1β changes to As4.1 cells before and after confluence. Cell lysates from cells grown to 70%–80% confluence in the presence of 10% FBS (A, lane 1), cells at day 2 postconfluence in the continuous presence of 10% FBS (A, lane 2) or in the absence of 10% FBS for two days after achieving 100% confluence (A, lane 3). After the aspiration of the incubation media, cells were lysed and centrifuged. Supernatant protein (60 μg) was resolved by SDS-PAGE on 7% or 15% acrylamide slab gels followed by Western blotting for N-cad, MRTF-A, sm MHC, nm MHC, and PP1β and then analyzed by densitometry. In the case of MRTF-A, a nuclear fraction was prepared, and 100 μg was resolved (B, upper panel). p-values were obtained by ANOVA. To assess nuclear localization of MRTF-A, cells were plated onto coverslips and fixed and permeabilized as described in the Methods section. Cells were then immunostained with primary MRTF-A antibody (1:100) and then with FITC-labelled goat anti-mouse secondary antibody to MRTF-A (1:1,000). Nuclei were stained with propidium iodide (DAPI) (B, lower panel). The density of preconfluent cells was arbitrarily set to 1. Fluorescence was analyzed by an image analyzer. Lane 1, preconfluent cells; lane 2, postconfluent cells in the presence of 10% FBS. p-values were obtained by unpaired Student’s t-tests (n = 4). Scale bar: 10 μM. (C) Postconfluent cells were incubated in DMEM + 10% FBS with or without CCG-1423 (5 × 10−6 M), an inhibitor of nuclear translocation of MRTF-A, for 1 h and then with ML-7 (6 × 10−5 M) for another 1 h. Secreted renin was measured by ELISA (C, n = 5). MRTF-A, myocardin related transcription factor-A; sm MHC, smooth muscle myosin heavy chain; nm MHC, nonmuscle myosin heavy chain; PP1β, protein phosphatase 1β; FBS, fetal bovine serum. NS, no significant difference. p-values within groups were obtained by paired Student’s t-tests, and those between groups were obtained by ANOVA.
Korean J Physiol Pharmacol 2022;26:479-499 https://doi.org/10.4196/kjpp.2022.26.6.479
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