Figure 2.

JAK1 and the ERK pathway cooperate to repress myogenic differentiation. (A) C2C12 cells stably expressing either a wild-type Flag-JAK1 or an empty vector (pcDNA3) were allowed to differentiate in DM for various times. 20 μg WCE was subjected to immunoblotting using various antibodies as indicated. (B) Duplicate C2C12 cells were cotransfected with MCK-luc together with either an empty vector or the wild-type Flag-JAK1. 24 h after transfection, cells were switched to DM for another 24 h before harvest. WCEs were subjected to luciferase analysis. Fold change was calculated as the ratio of the luciferase activity of cells transfected with Flag-JAK1 over that with an empty vector. The results are presented as mean ± SD (error bars). (C and D) C2C12 cells were transfected with either cDNA expression vectors (C) or various siRNAs (D) as indicated. (C) Cells were grown in GM for 24 h and in DM for another 24 h. (D) 10 μM DMSO or U0126 was added when cells were switched to DM and kept for 12 h. Cells were harvested, and WCEs were subjected to immunoblotting. + denotes that cells were transfected with the construct indicated on the left; ca, constitutively active. The white line indicates that intervening lanes have been spliced out.