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. 2020 Sep 25;11:566584. doi: 10.3389/fphys.2020.566584

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Copyright © 2020 Pirkmajer, Bezjak, Matkovič, Dolinar, Jiang, Miš, Gros, Milovanova, Pirkmajer, Marš, Kapilevich and Chibalin.

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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Ouabain stimulates the phosphorylation of S6RP in cultured human myotubes. (A–F) Human myotubes were treated with or without 50 nM ouabain (OUA) in Advanced MEM with 2% FBS for 20 h and in serum-free Advanced MEM for 4 h. Cells were treated with tocilizumab (100 μg/ml, TCB) during the last 4 h and with interleukin-6 (50 ng/ml, IL-6) during the last 15 min. (G–R) Human myotubes were treated in serum-free Advanced MEM with different concentrations of OUA for 24 h (G–L) or with 50 nM OUA for 1–24 h (M–R). (A,G,M) The total S6RP, (B,H,N) phospho-S6RP (Ser235/236), (D,J,P) the total 4E-BP1, and (E,K,Q) phospho-4E-BP1 (Thr37/46) were measured by immunoblotting. Graphs in panels (C,I,O) show the phospho-S6RP:S6RP ratio and graphs in panels (F,L,R) show the phospho-4E-BP1:4E-BP1 ratio. Results are means with SEM (n = 4–8). *p < 0.05 vs. Basal.