Figure 4. Quantification of MLCK levels in guinea-pig ileum and the effect of [ATP] on inhibition of MLCK by HA1077.
A, guinea-pig ileum longitudinal smooth muscle proteins were extracted as described in Methods, subjected to SDS-PAGE and immunoblotted with anti-MLCK. Three loading levels of three independent extracts and different known amounts of purified chicken gizzard MLCK (0.05, 0.1, 0.15, 0.2, 0.4 and 0.8 μg) were applied to the gel. MLCK bands were quantified by densitometric scanning. A standard curve relating the intensity of the chicken gizzard MLCK bands (optical density × area of the band) to micrograms of MLCK loaded was linear with an r2 value of 0.98. B, the effect of increasing concentrations of HA1077 on the activity of purified MLCK was assayed as described in Methods at 0.25 (○) and 1 mM ATP (•).