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. 2019 Apr 3;13:21. doi: 10.3389/fncir.2019.00021

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Copyright © 2019 Autuori, Sedlak, Xu, Ridder, Tedoldi and Sah.

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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rSK1 regulates the membrane expression of rSK2 channels. (A) Immunocytochemistry of Cosm6 cells transfected with rSK2-Myc or rSK1-HA. rSK2-Myc (pink), rSK1-HA (green), DAPI (blue). (B) Immunoblots from Cosm6 cells transfected with rSK1-HA, rSK2-Myc and hSK1 in different combinations. Shown are the total lysate (LYS) and membrane (MEMB) fraction isolated using surface biotinylation probed with either Myc (rSK2; left) or HA (rSK1; right) antibody. EGFR was used as the loading control. rSK2 transfection results in three size bands that represent monomeric protein (band 3, ~60 kDa), dimers (band 2, ~120 kDa) and tetramers (band 1, >200 kDa). Co-transfection of rSK2 and rSK1 reduced the amount of rSK2 expressed in the cell membrane. (C) Bar graphs show normalized relative density of SK2, as described in the “Materials and Methods” section, in the membrane fraction from Cosm6 cells transfected with rSK2 alone or co-transfected with rSK1 or hSK1. Co-transfection with rSK1-HA significantly reduced the 60 kDa band (p = 0.002). Co-transfection with hSK1 increased expression of rSK2. (D) Co-immunoprecipitation (co-IP) assay of transfected Cosm6 cells shows co-assembly of rSK1-rSK2. Solubilized protein from Cosm6 cells transfected with rSK1 and/or rSK2 was immunoprecipitated with Myc (left) or HA (right) and immunoblotted with either Myc or HA as indicated.