Fig. 6

Timeless binds to Myosin-9 to maintain its stability. (a) Myosin-9 protein was identified by mass spectrometry. (b) Immunoprecipitation analyses of the interaction between Timeless and Myosin-9 in HCT116 cells. (c) Immunofluorescence costaining of Timeless (Red) and Myosin-9 (Green) to indicate colocalization in HCT116 (upper) and RKO (lower) cells. Original magnification, × 400. Western blotting was performed to detect total (d) and subcellular Myosin-9 (e) in Timeless knockdown and overexpressing CRC cells. GAPDH and Histone-3 were used as internal loading controls, respectively. (f) CRC cells with specific Timeless siRNA or control siRNA were treated with 100 μg/ml CHX and harvested at different time points as indicated. Myosin-9 was detected by western blotting and quantified by densitometry. (g) Timeless knockdown and control CRC cells were treated with 5 μM of MG132 for 12 h, and Myosin-9 protein was detected by western blotting. Error bars indicate the means ± SD. *P < 0.05, **P < 0.01