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. 2019 Feb 4;10:59. doi: 10.3389/fphar.2019.00059

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Copyright © 2019 Palikhe, Ohashi, Sakamoto, Hattori, Kawakami, Andoh, Yamazaki and Hattori.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effect of GRK2 siRNA transfection on TLR3-mediated signaling for iNOS expression in MG6 cells. (A) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then the time course of changes in IRF1 levels in each fraction after 50 μg/ml poly(I:C) application was tracked by Western blot analysis. (B) Effect of transfection of GRK2 siRNAs (siGRK2) on nuclear translocation of IRF1 60 min after challenge with 50 μg/ml poly(I:C) was compared with that when the negative control siRNAs (siCTL) was transfected. GAPDH and lamin B served as a cytoplasmic and nuclear marker, respectively. (C) Effect of siGRK2 transfection on IFN-β mRNA expression levels 3 h after 50 μg/ml poly(I:C) application. (D) Effect of siGRK2 transfection on phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 4 h after 50 μg/ml poly(I:C) application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (E) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 50 μg/ml poly(I:C) application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ^∗P < 0.05 by t-test.