HEK293 cells stably expressing FLAG-tagged β2AR were either directly stimulated for 5 min with the βAR agonist ISO or different β-blockers at indicated concentrations (A) n = 4), or pretreated for …
Excel spreadsheet containing the individual numeric values of phosphorylated β2AR / total β2AR relative density analyzed in Figure 1.
HEK293 cells stably expressing FLAG-tagged β2AR were treated with increasing concentrations of CAR (A) n = 4) and ALP (B) n = 3), or pretreated for 15 min with 10 μM β2AR antagonist ICI118551 (C) n =…
Excel spreadsheet containing the individual numeric values of phosphorylated β2AR / total β2AR relative density analyzed in Figure 2A-D.
HEK293 cells stably expressing FLAG-tagged β2AR were stimulated by isoproterenol (ISO) or carvedilol (CAR) for 30 min with indicated concentrations. Cell lysates were analyzed for PKA-phosphorylated …
HEK293 cells stably expressing FLAG-tagged β2AR were stimulated by 1 μM isoproterenol (ISO) or carvedilol (CAR) for indicated times. Cell lysates were analyzed for PKA-phosphorylated β2AR, …
(A) HEK293 cells co-expressing FLAG-tagged β2AR, HA-tagged Gsα and EGFP were stimulated with 100 nM ISO or indicated β-blockers for 5 min. In proximity ligation assay (PLA), cells were …
Excel spreadsheet containing the individual numeric values of PLA dots / cell number in each raw image analyzed in Figure 3A, and the individual numeric values for maximum FRET responses in Figure 3B-E.
HEK293 cells stably expressing FLAG-tagged β2AR were pretreated with the Gi inhibitor pertussis toxin (PTX, 200 ng/ml, 16 hr) or the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (ddA, 50 μM, …
The cAMP biosensor ICUE3 and FLAG-β2AR were co-expressed in HEK293 cells. Cells were treated with 100 nM βAR agonist ISO or different β-blockers, and changes in cAMP FRET ratio were measured. In …
HEK293 cells expressing either CAAX-ICUE3 targeted to non-rafts regions of the plasma membrane (A) or LYN-ICUE3 targeted to rafts regions of the plasma membrane (B) were treated with 1 μM ISO or …
(A) Rat neurons on 10–14 days in vitro (DIV) were treated for 5 min with 1 μM indicated drugs. The phosphorylation of endogenous LTCC α11.2 subunit was determined with phospho-specific antibodies, …
Excel spreadsheet containing the individual numeric values of phosphorylated a11.2 or GluA1 / total a11.2 or GluA1 relative density analyzed in Figure 4.
(A) Representative single channel recordings of LTCC CaV1.2 currents using 110 mM Ba2+ as charge carrier in rat hippocampal neurons on 7–10 days in vitro (DIV) after depolarization from −80 (hp) to …
Excel spreadsheet containing the individual numeric values of Po, availability and current analyzed in Figure 5C-G.
Cells were depolarized with 110 mM Ba2+ for 2 s from a holding potential of −80 mV to 0 mV and the NPopen was determined over time. (A) Diagram reflecting our patch statistics without using BayK in …
Rat cortical neurons on 7 days in vitro (DIV) were incubated overnight in fresh medium with or without 2 mM Ca2+ (no Ca2+), cells were then either mock treated (NT), or treated with 1 μM indicated …
(A) Schematic of an engineered β2AR with S204/207A double serine mutations that loses high affinity binding to ISO but not CAR at nanomolar range. (B) cAMP biosensor ICUE3 and β2AR wild-type (WT) or …
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 6B, and the individual numeric values of phosphorylated β2AR / total β2AR relative density analyzed in Figure 6C-D.
(A–B) β1AR/β2AR double knockout (DKO) mouse hippocampal neurons on 7–10 days in vitro (DIV) were cotransfected with FLAG-tagged β2AR WT (A) or mutant (B) and HA-tagged LTCC α11.2 subunit, 24 hr …
Excel spreadsheet containing the individual numeric values of Po, availability and current analyzed in Figure 7E-G.
β1AR/β2AR double knockout (DKO) hippocampal mouse neurons at 7–10 days in vitro (DIV) were cotransfected with FLAG-tagged β2AR WT (A) or mutant (B) and HA-tagged LTCC α11.2 subunit, 24 hr later …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain (Mus musculus) | β1AR/β2AR double knockout | Jackson Laboratories | Stock # 003810 | |
Strain (Rattus norvegicus) | Sprague Dawley | Charles River Laboratories | ||
Cell line (Homo sapiens) | HEK293/β2AR-WT | De Arcangelis et al., 2009 | HEK293 cells stably expressing FLAG-β2AR | |
Cell line (Homo sapiens) | HEK293/β2AR-S204/207A | This paper | HEK293 cells stably expressing FLAG-β2AR-S204/207A | |
Antibody | Phospho-β2AR (Ser261/262) (mouse monoclonal) | Dr. Richard Clark (UT Huston) | Clone 2G3 | IF (1 μg/ml), WB (1:1000) |
Antibody | Phospho-β2AR (Ser355/356) (mouse monoclonal) | Dr. Richard Clark (UT Huston) | Clone 10A5 | WB (1:1000) |
Antibody | β2AR (rabbit polyclonal) | Santa Cruz Biotechnology | sc-570 RRID:AB_2225412 | PLA (1:100), WB (1:1000) |
Antibody | Phospho-β2AR (Ser355/356) (rabbit polyclonal) | Santa Cruz Biotechnology | sc-16719R RRID:AB_781609 | WB (1:1000) |
Antibody | α11.2 (rabbit polyclonal) | Patriarchi et al., 2016 | FP1 | WB (1:1000) |
Antibody | Phospho-α11.2 (Ser1928) (rabbit polyclonal) | Patriarchi et al., 2016 | CH3P | IF (1 μg/ml), WB (1:1000) |
Antibody | Phospho-α11.2 (Ser1700) (rabbit polyclonal) | Patriarchi et al., 2016, Originally from Dr. William Catterall (U of Washington) | WB (1:1000) | |
Antibody | GluA1 (rabbit polyclonal) | Patriarchi et al., 2016 | WB (1:1000) | |
Antibody | Phospho-GluA1 (Ser831) (rabbit polyclonal) | Patriarchi et al., 2016 | WB (1:1000) | |
Antibody | Phospho-GluA1 (Ser845) (rabbit polyclonal) | Patriarchi et al., 2016 | WB (1:1000) | |
Antibody | FLAG-M1 | Sigma-Aldrich | F3040 RRID:AB_439712 | IF (1 μg/ml), WB (1:1000) |
Antibody | HA | Covance | MMS-101R RRID:AB_291262 | PLA (1:1000) |
Recombinant DNA reagent | β2AR-mutant | This paper | FLAG-tagged human β2AR with S204/207A double mutations | |
Recombinant DNA reagent | β2AR-ICUE3 | This paper | ICUE3 fused to the C-terminal of human β2AR | |
Commercial assay or kit | Duolink in situ detection reagents | Sigma-Aldrich | DUO92007 | PLA |
Software, algorithm | pCLAMP10 | Molecular Devices | electrophysiology | |
Software, algorithm | MetaFluor | Molecular Devices | FRET |
Biophysical properties of L-type Ca2+ currents in the neurons recorded in Figure 5A–5E.
Values are mean ± SEM. *p<0.05 with Kruskal Wallis – Dunn’s multiple comparison test.
Biophysical properties of L-type Ca2+ currents in the neurons recorded in Figure 7C–7G.
Values are mean ± SEM. *p<0.05 with Kruskal Wallis – Dunn’s multiple comparison test.