Anti p44 42 erk

Serum-starved transfected HEK293 cells were stimulated with increasing concentrations of calcium chloride (CaCl 2 ; 0.5, 1.0, 3.0 and 5.0 mM) with or in the absence of the calcimimetic R568 (0.01 μM; kindly provided by Amgen Inc.) for 10 min at 37°C in saline solution PSS (NaCl 125 mM, KCl 4 mM, HEPES 20 mM, d-Glucose 0.1%, NaH 2 PO 4 0.8 mM, MgCl 2 1 mM, pH 7.45). Incubation was stopped by placing the cells on ice. The PSS was removed and the cells were treated with 50 μL ice-cold lysis buffer as described previously, supplemented with complete phosphatase inhibitor cocktail (Roche Diagnostics Spa). Samples (20 μg proteins/well) were denatured with loading dye and β-mercaptoethanol for 10 min at 95°C. Proteins were separated on 10% w/v SDS-PAGE and analysis of ERK1/2 activation was performed by western blot with 1:1000 and 1:2000 dilution polyclonal anti-p44/42 ERK and anti-phospho-p44/42 ERK antibodies, respectively (Cell Signaling Technology). Specific protein bands were detected by a chemiluminescent method as described previously for CASR and FLNA proteins quantification. Experiments were repeated at least thrice.