Xenbase Image ID: 152321

Fig. S7. Characterization of mTORC1 signaling. (A and B) Western blot analysis of Lewis lung carcinoma-porcine kidney 1 (LLC-PK1) cells treated with different concentrations of Insulin (A) or Igf2 (B) for 20 min using pS6 as readout for mTORC1 activity. (C and D) pS6 Western blot analysis of LLC-PK1 cells treated with 100 nM Insulin (C) or 0.01 nM recombinant Igf2 (D) for 0–60 min. Note that the maximal activation occurs between 20 and 40 min. α-Actin was used as a loading control. (E–J) pS6 immunofluorescence analysis of human HK2 cells in the presence or absence of 0.1 μM Insulin or 0.01 nM Igf2 comparing cells lipofected with a scrambled MO (scr-MO) or sDicer-MO. Note that the sDicer-MO perfectly matched the human Dicer sequence and thus could be used to inhibit the translation of the human protein. (K–N) pAkt immunofluorescence analysis comparing the proximal tubules of xTsc1, xDicer, and xTsc1/xDicer double morphants to those of uninjected control Xenopus embryos. (O) Luciferase assays of LLC-PK1 cells transfected with the empty vector, pmirGLO-Tsc1, or pmirGLO-Tsc1-mut alone or in combination with lipofected sDicer-MO plus/minus a mixture of miR-19b and miR-130b mimics. The graph is an average of three independent experiments. SD is indicated in the individual bars. Note that deletion of the miR-19b/miR-130b binding sites had only minimal effect on the activity of the reporter probably due to the length of the 3′ UTR and the abundance of other microRNA (miRNA) binding sites. Image published in: Romaker D et al. (2014) Copyright © 2014. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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