Anti p38 mapk antibody

These assays were carried out as described elsewhere. ^{(21 (link))} Briefly, cells were incubated for 10-mins in either Experimental Buffer (0.5mM Ca²⁺_o) alone or in buffer supplemented with either a) various concentrations of Ca²⁺_o (2 – 10mM), or b) various concentrations of NPSP795 (1–1000nM; equal DMSO concentration in all samples) in the presence of either 2.4 mM Ca²⁺_o (ADH mutants) or 3.4 mM Ca²⁺_o (Wild-type); these Ca²⁺_o being close to ~EC₈₀ values for these receptors (with regards to ERK activation). Where [CaCl₂] was increased, [NaCl] was reduced accordingly to normalize ionic strength. Where NPSP795 was used, cells were pre-exposed to the calcilytic for 30-secs before application of the high Ca²⁺_o/NPSP795 co-treatment. The cell lysate supernatant was stored at −80°C until use, whereupon an aliquot was mixed with 5X Laemmli buffer and boiled for 3-min prior to immunoblotting (40–60 μl sample/lane) using the phospho-specific anti-ERK antibody or anti-p38^MAPK antibody (1:5000; Cell Signaling). Protein equivalency of the samples was demonstrated initially by Ponceau staining the blot (prior to blocking) and then by stripping and reprobing with either total anti-ERK or anti-β-actin antibodies. Relative immunoreactivity was determined by densitometry.