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. 2019 Jun 5;10(1):186–194. doi: 10.1016/j.apsb.2019.05.005

Figure 4.

Figure 4

IGF-1 regulates OAT3 phosphorylation through PKA pathway. (A) Top panel: OAT3-expressing cells were lysed, pre-cleared and immunoprecipitated with control IgG-agarose (as negative control) or anti-myc agarose affinity gel, followed by immunoblotting (IB) with anti-phospho-Ser/Thr antibody. Bottom panel: The identical blot of the top panel was re-probed with anti-myc antibody to determine the amount of OAT3 immunoprecipitated. (B) Phosphorylation of OAT3. Top panel: OAT3-expressing cells were treated with IGF-1 (100 nmol/L) with or without PKA inhibitor H89 (10 μmol/L) for 4 h. After treatment, cells were lysed, pre-cleared and immunoprecipitated with anti-myc agarose affinity gel, followed by immunoblotting (IB) with anti-phospho-Ser/Thr antibody. Bottom panel: The identical blot of the top panel was re-probed with anti-myc antibody to determine the amount of OAT3 immunoprecipitated. (C) Densitometry analysis of blot results from Fig. 4B, top panel as well as from other experiments. The values are mean ± SD (n = 3); *P < 0.05. Statistical analysis was performed using one-way ANOVA by using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).