Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots
Figure 2
Antibody specificity using western blots.
A. Full length western blots with single lanes loaded with 25 µg of protein/lane demonstrating the relevant bands (*) used for quantification in other experiments. Antibodies against GAPDH, total MLC20, PMLC20 and diphospho-MLC20 (PPMLC20) each identified one prominent band, though faint “non-specific” bands appeared with the anti-GAPDH and anti-PPMLC20 antibodies. B. Western blots performed as in panel A but with the addition of phos-tag (30 µM) to the gel matrix to promote mobility shifts in phosphorylated proteins. Probing for total MLC20 reveals 3 bands corresponding to unphosphorylated (0 P), mono-phosphorylated (1 P), and diphosphorylated (2 P) MLC20. The anti-PMLC20 antibody reacts with mono- and diphosphorylated MLC20 and fails to recognize the unphosphorylated species. The anti-PPMLC20 antibody recognizes primarily diphosphorylated MLC20 and demonstrates almost no cross-reactivity with the unphosphorylated or mono-phosphorylated MLC20. Molecular weight markers (MW) are shown to the left of each blot.