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. 2011 Dec 15;6(12):e29173. doi: 10.1371/journal.pone.0029173

Figure 3. miR-133 negatively regulates the IGF-1R signaling pathway in C2C12 cells.

Figure 3

(A) C2C12 cells were transfected with negative control or miR-133 mimics at 50 nM or 100 nM for 36 hours. (B) C2C12 cells were transfected with 50 nM RNA mimics and inhibitors as indicated for 36 hours. (C) C2C12 cells were transfected with 50 nM negative control, miR-133 mimics or anti-IGF-1R siRNA. Proteins were extracted for western blotting against IGF-1R. α-tubulin was served as loading control. The mRNA levels of IGF-1R were determined by semi-quantitative RT-PCR, and GAPDH was used as internal control. (D) C2C12 cells were transfected as in (C). 24 hours after transfection, cells were serum starved for 24 hours and incubated in differentiation medium supplemented with 5 nM IGF-1 for 30 minutes. Proteins were extracted for western blotting against Ser-473 phosphorylated Akt and total Akt. GAPDH was used as a loading control. Representative results from independent experiments (n≥2) are shown. The numbers below the blots represent relative expression levels.