Anti β1ar ab3442

Immunoblotting was performed on cell extracts according to methods described previously or manufacturer’s instructions^{55 (link)}. The dilutions for primary and secondary antibodies were as follows: Abcam anti-β₁AR (ab3442) at 1:3000 followed by secondary goat anti-rabbit IgG at 1:5000; Santa Cruz anti-β₁AR (clone V-19) at 1:1000 followed by secondary goat anti-rabbit IgG at 1:2000; anti-HA (Y-11) at 1:3000 followed by secondary goat anti-rabbit IgG at 1:5000; anti-Flag M2 at 1:700 followed by secondary goat anti-mouse IgG at 1:1000; anti-pERK at 1:2000 followed by secondary goat anti-rabbit IgG at 1:3000; anti-ERK at 1:3000 followed by secondary goat anti-rabbit IgG at 1:5000; anti-β-actin at 1:2500 followed by secondary goat anti-mouse IgG at 1:4000. Each panel in each figure represents the results from a single gel (exposed for a uniform duration); detection was with enhanced chemiluminescence or LI-COR Odyssey CLx imaging system (LI-COR Biosciences) with image Studio Lite Ver 5.0 software used for quantification of protein expression. All results were replicated in at least three experiments on separate culture preparations.

Antibodies were from the following sources: anti-β₁AR (clone V-19, which was raised against a peptide that maps to the C-terminus of the mouse β₁AR) and anti-HA (clone Y-11) were from Santa Cruz Biotechnology (Dallas, TX). Anti-β₁AR (ab3442, raised against residues 394–408 in human β₁-ARs) and anti-β-actin were from Abcam (Cambridge, MA). Anti-Flag M2 antibody was from Sigma-Aldrich (Saint Louis, MO). Antibodies that recognize ERK protein and phosphorylation were from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit and goat anti-mouse IgG (H + L)-Horse radish peroxidase conjugates were from Bio-Rad Laboratories, Inc. (Hercules, CA). Peptide-N-glycosidase F (PNGF), O-glycosidase (O-Gly), neuraminidase (Neu), propranolol, isoproterenol (Iso), and tunicamycin were obtained from Sigma-Aldrich (Saint Louis, MO). All other chemicals were reagent grade.