β-Adrenergic receptor density and internalization assays.

β-Adrenergic receptor density was measured by the methods of Maisel et al. (35) with modifications. Sarcolemmal membrane and light vesicle fractions were prepared from the LV. The total β-adrenergic receptor density was determined as the amount of bound radioligand [³H]CGP-12177 (PerkinElmer, Waltham, MA). Filtered radioactivity was counted in a liquid scintillation counter (Beckman), and data were analyzed with a GraphPad Radioactivity Calculator (La Jolla, CA).

β-Adrenergic receptor internalization was evaluated as described previously (34) with modifications. Briefly, HEK-293 cells were plated in complete DMEM and transiently transfected with β₁-adrenergic receptor and β-arrestin 1 (as a positive control), Txnip, or empty vector (as a negative control). Cells were then incubated in ¹²⁵I-cyanopindolol (PerkinElmer) at 37°C for 5 min. Incubations were stopped by placing the cells on ice and rapidly washing twice with ice-cold PBS. The cells were kept on ice for 10 min in acid wash solution (150 mM NaC1 and 50 mM acetic acid) to remove the surface-bound radioligand. The supernatant containing the acid-released radioactivity was collected, and the cells were treated with 0.5 M NaOH and 0.05% SDS to solubilize the acid-resistant (internalized) radioactivity. Radioactivity was measured, and the percent internalization at each point was calculated from the ratio of the acid-resistant binding to the total binding.