MyHC is selectively targeted by TNF/IFN signaling. C2C12 myoblasts were differentiated in DM for 3 days and subsequently switched to medium alone or containing TNF (10 ng/ml) and IFN (100 U/ml) for 48 hours. (A) Immunofluorescence to detect expression of myofibrillar proteins (MyHC, fast twitch; troponin T [Tn]; tropomyosin, α and β [TM]; actin, α skeletal). Images are shown at ×20 magnification. (B) Immunostained expression levels of myofibrillar proteins were quantitated using Zeiss AxioVision software (Carl Zeiss Inc., Thornwood, New York, USA). The data were calculated from a minimum of ten randomly chosen fields of cells at ×4 magnification. (C) Whole-cell extracts were prepared from untreated or TNF/IFN–treated myocytes, and 50 μg total protein was used in Western analyses to probe for myofibrillar proteins (troponin; α- and β-tropomyosin; MyLC, myosin light chain). (D) Immunofluorescence of untreated (black bars) or TNF/IFN–treated (hatched bars) C2C12 myotubes, probing for MyHC (Ab1, MyHC antibody MY-32; Ab2, MyHC antibody MF20). (E) Primary myotubes were either untreated (black bars) or treated with TNF and IFN (hatched bars) for 48 hours. Cells were stained by immunofluorescence for myofibrillar proteins, and the level of protein expression was quantitated by digital capture and AxioVision software. For histograms, each bar represents mean ± SEM from three independent experiments.