Anti ha agarose antibody beads

HEK293 cells stably expressing hemagglutinin (HA)-tagged PTHR and cultured on a 10-cm dish were stimulated with PTH(1–34)-NH2 or analogues at 100 nM for 5 min. Cells were then washed with ice-cold PBS prior to crosslinking for 2 h with dithiobis (succinimidyl propionate) DSP (Covachem, #13301) in PBS at 4 °C. The reaction was stopped by addition of 10 mM Tris–HCl for 10 min and cell lysates were prepared using lysis buffer (1% Triton X-100, 50 mM Tris–HCl pH 7.4, 140 mM NaCl, 0.5 mM EDTA) containing protease and phosphatase inhibitors (Roche, #11873580001). Protein concentration was determined using BCA protein assay kit (ThermoFisher, #23225), and lysates were incubated with anti-HA agarose antibody beads (Sigma-Aldrich; #A2095 clone HA-7) overnight at 4 °C. Elution was done using LDS loading buffer (Life Technologies) and samples were loaded on 10% SDS-PAGE and transferred to nitrocellulose membrane. We used primary antibodies against HA (Covance, clone 16B12, Mouse IgG1) and β-arrestin1/2 (Cell Signaling; #4674, clone D24H9, Rabbit IgG); anti-Mouse HRP and anti-Rabbit HRP (Dako, Goat polyclonal) secondary antibodies were then used. Immunoreactive bands were visualized with Luminata Forte (EMD Millipore) and autoradiography film.