Figure. 2.

An in vitro model system to express RPL3L in C2C12 myogenic cells. A. Schematic of the pINDUCER Tet-On system used to express of RPL3L in C2C12 cells. Upon doxycycline (Dox) binding, constitutively expressed rtTA becomes activated with subsequent binding to the tetracycline response element (TRE), thereby inducing expression of HA-tagged RPL3L. B. Western blot showing exogenous RPL3L expression in myotubes in response to Dox treatment. RPL3L-C2C12 cells were differentiated four days in differentiation media containing 1 μg·mL⁻¹ Dox with RPL3L expression detected using a HA antibody. Alpha-tubulin was used as a loading control for Western blot analysis. C. Immunostaining showing the presence of exogenous RPL3L in myotubes in response to Dox treatment. RPL3L-C2C12 cells were differentiated four days in differentiation media with or without Dox (1 μg·mL⁻¹) and incubated with an antibody against HA. Nuclei were labelled by DAPI staining. D–E. RNA concentration and electrophoresis showing 18S and 28S rRNA following RPL3L-HA immunoprecipitation from 4-day differentiated myotubes treated with or without Dox (1 μg·mL⁻¹). F. Immunoblotting for exogenous RPL3L and RPS6 proteins from protein lysates and immunoprecipitate samples. TRAP: translating ribosome affinity purification.