Fig. 2

MYL6B negatively regulates p53 by promoting its’ ubiquitination. (a) Huh7 was processed for immunofluorescence analysis with anti-MYL6B (a-c). Huh7 transfected with Myc-DDK-MYL6B was subjected for immunofluorescence analysis with anti-FLAG and combing with either anti-p53 (d-i) or anti-MDM2 (j-l). DAPI (4′,6-diamidino-2-phenylindole) was used to label nuclei. (b) Huh7 was cotransfected with FLAG-p53, MYL6B and myc3-MDM2. Forty-eight hours later, cells were treated with 20 μM MG132 for 4 h and harvested for immunoprecipitation assay with anti-FLAG Agarose Affinity Gel. The eluted proteins were then subjected to immunoblot analysis with ubiquitin, p53, MYL6B and MDM2 antibodies. (c) Increasing amounts (0, 1, and 2 μg) of MYL6B plasmid were transfected into Huh7 (upper panel) and SK-HEP-1 (lower panel) alone or together with MDM2 plasmid, and followed by immunoblotting with p53, Bax, MDM2, MYL6B and GAPDH antibodies. (d) Huh7 transfected with increasing amounts (0, 1, and 2 μg) of MYL6B plasmid was treated with blebbistatin (4 μM), latrunculin A (2 μM) or vehicle and then subjected to immunoblot analysis with p53, Bax, MDM2, MYL6B and GAPDH antibodies