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. 2019 Aug 13;14(8):e0216167. doi: 10.1371/journal.pone.0216167

Fig 3. RGS12 interacts with activated H-Ras and is downregulated within the C2C12 cell line during differentiation.

Fig 3

(A) Immunoblotting (IB) of cell lysates from three stable clones of the C2C12 cell line indicating their stable expression of a constitutively-active, GTPase-deficient (G12V) mutant of H-Ras protein (via detection of its N-terminal myc-epitope tag). (B) Co-immunoprecipitation of myc-tagged, activated H-Ras (G12V mutant) with endogenous RGS12 protein expressed in the C2HRas cell line clone 9. IP: immunoprecipitation; IB: immunoblotting. (C) To test the effect of stable expression of activated H-Ras on myoblast differentiation in vitro, multi-nucleated myotube content of indicated cell populations (either parental C2C12 cells [panels i, ii] or C2HRas clone #9 cells [panels iii, iv]) was measured by fixation and staining for sarcomere myosin (MHC; green) and nuclear DNA content (DAPI; pseudocolored red), either pre-differentiation (panels i, iii) or post-differentiation by culture for 5 days in low serum (2% horse serum; panels ii, iv). The mean fusion index for several C2C12 cell populations was 40% (± 2.5%; SEM), consistent with other reports [68, 69]. In contrast, the C2HRas cell line clone 9 was incapable of myotube formation (panel iv) and no fusion index could be calculated. (D) Co-immunoprecipitation of myc-tagged, activated H-Ras (G12V mutant) with ectopically co-expressed, HA-tagged RGS12 in C2C12 cells. IgH: immunoglobulin heavy-chain. (E) Endogenous levels of RGS12 protein are down-regulated during C2C12 differentiation into myotubes (i.e., 7 days of culture in low serum medium), whereas the same culture conditions did not lower RGS12 levels within the C2HRas cell line clone #9. (F) Mammalian RGS12 proteins encode a “destruction box” recognition motif, conforming to the consensus RxxLxxxx(D/N), where “x” is any amino-acid, that is found in most substrates of the Anaphase-Promoting Complex (APC). This destruction box sequence is completely conserved across human (h), mouse (m), and rat (r) RGS12 proteins (i.e., amino acids 402–410 of mouse RGS12: RAFLDGDAD); this consensus motif is preserved in zebrafish (z) and Drosophila (d) RGS12 orthologs, as well as in the APC targets Skp2 and Myf5.