Tyr416

Cultured mouse DRG neurons were treated with 20 nM PTHrP or 20 nM PTHrP with 100 nM PP2 or vehicle control for 10 min at 37°C and then lysed with lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1 mM sodium fluoride, 1 mM phenylmethanesulfonyl fluoride and 1× Protease Inhibitory Cocktail (leupeptin, aprotinin, antipain, and benzamidine-HCl). Lysates were run on a 7.5% SDS-PAGE gel and then transferred to nitrocellulose membranes. Following blocking in 4% non-fat milk in Tris-buffered saline, membranes were probed with either rabbit polyclonal anti-Src (1:1000, Clone 36D10, Cell Signaling, Danvers, MA) or rabbit polyclonal anti-Phospho-Src (1:1000, Tyr416, Cell Signaling) antibodies. Membranes were then washed and incubated with goat anti-rabbit IgG-HRP secondary antibody (1:5000, Antibodies Inc., Davis, CA). Blots were incubated with ECL-Plus reagent (Perkin Elmer, Waltham, MA), and the chemiluminescence signals were captured on X-ray film (Kodak-Biomax, Carestream Health, Rochester, NY). Experiments were repeated in four batches of cultured mouse DRG neuron lysates. Signal intensities of pSrc bands were normalized to the signal intensities of tSrc bands for each individual treatment groups using NIH ImageJ software, and presented as fold-change in pSrc levels upon PTHrP or PTHrP+PP2 treatment conditions.