FIGURE 5.

Identification of specific PP2A complexes in regulation of MT expression. A, immunoblotting analysis of MT expression in HEK cells expressing shRNA that targets GFP or expressing two independent shRNAs that target B56β or PR110 with the treatment of 40 μm CdCl₂ or 100 μm ZnSO₄. A MTT assay was performed to measure the cytotoxicity at the indicated concentrations. The corresponding cell viability is indicated under each lane. *, p < 0.05 compared with the SHGFP control cells. B, HA-tagged MTF-1 was co-expressed with shRNAs targeting GFP, B56β, and PR110 in 293FT cells and followed by a treatment of 40 μm CdCl₂ for 12 h. 3 mg of the cell lysates were co-immunoprecipitated with an antibody against the HA tag and followed by immunoblotting analysis with specific antibodies indicated. The lower panel shows the input of each protein indicated corresponding to the IP assay. The value under each band indicates -fold change of p-MTF-1 level normalized to HA-tag expression relative to SHGFP cells. C, 293FT cells were transfected with vectors encoding HA-tagged MTF-1 for 48 h and treated with 40 μm CdCl₂ or 100 μm ZnSO₄ for 6 h before harvesting. 3 mg of the cell lysates was subjected to co-IP using antibodies against the HA tag and PP2Ac and followed by immunoblotting using antibodies against B56β, PR110, PP2Ac, and the HA tag. D, Myc-tagged MTF-1 was co-expressed with HA-tagged vectors encoding B56β or PR110 in 293FT cells for 48 h and followed by a treatment of 40 μm CdCl₂ for 6 h. The co-IP assay was performed with an antibody against the HA tag and followed by immunoblotting using specific antibodies against the myc tag and the HA tag.