Figure 1.

Efficient internalization and recycling of the β₂AR in HEK293 cells simultaneously depleted of NHERFs 1 and 2. (A) Schematic of NHERF1 (also called EBP50) and NHERF2 (also called E3KARP) domain organization, showing PDZ domains and ERM protein-binding domain (EBD). (B) Verification of dual NHERF1 + 2 knockdown by immunoblotting. Arrows indicate specific bands; nonspecific bands (NS) verify equal loading between the indicated samples. Molecular mass markers (in kilodaltons) are indicated. Images shown are representative of three experiments. (C) Flow cytometric assessment of receptor internalization and recycling. HEK293 cells stably expressing either FLAG-tagged, wild-type β₂AR, or the FLAG-β₂AR-Ala PDZ mutant were transfected with the indicated siRNAs and assayed for surface receptor immunoreactivity before and after an agonist pretreatment and washout using fluorescence flow cytometry. (D) Recycling efficiency calculated from data shown in C, as described in Materials and methods. All error bars indicate SEM. P-values: Student’s t test with the negative control condition; n = 3 or 4.