Figure 6. PP1Ba and PP1Bb dephosphorylate the regulatory myosin light chain.

HeLa cells were transfected with zebrafish Mypt1 and GFP-PP1Ba (A, B) or zebrafish Mypt1 and GFP-PP1Bb (C, D). The HeLa cells were immunostained using an anti-phospho myosin light chain 2 antibody, co-stained with DAPI and imaged using confocal microscopy. Panels A and C show anti-phospho MLC2 staining, while B and D show a merge of GFP (green), phospho-MLC2 (red) and DAPI (blue). (E) Purified GST-MLC2 was run either as an untreated control (MLC2) or phosphorylated using ZIPK (Phosph-MLC2). The prephosphorylated MLC2 was dephosphorylated by Mypt1 (1-300)-PP1Ba or PP1Bb complexes immunoprecipitated from HEK293T cell lysates or using controls of treatment with beads from a myc-IP from untransfected cells (myc IP) or cell expressing only Mypt1 (1-300) and no additional catalytic subunit (Mypt1). Dephosphorylation (0), mono (1) and di-phosphorylation (2) was detected by band shift using a phos-tag SDS-PAGE gel as described in the materials and methods. HeLa cells were grown on fibronectin coated coverslips and treated with media containing either 0.1% DMSO (F) or 50 µM blebbistatin (G) for 4 hours. After treatment the HeLa cells were stained with DAPI (blue) and Alexa 568-phalloidin (red) and imaged with confocal microscopy and a color merged image is shown.