Fig. 3.

Overexpression of AGS3 modestly affects the internalization or recycling of Kir2.1 at later, but not earlier, time points when compared with control. Surface proteins of COS7 cells overexpressing Kir2.1 and either AGS3 or pcDNA3 (control) were labeled by using the membrane-impermeant Sulfo-NHS-SS-Biotin (Pierce) and allowed to internalize for a period of 0, 5, 10, or 20 min. Remaining surface biotin was cleaved by using a membrane-impermeant reducing agent, mercaptoethanesulfonic acid (MESNA, 50 mM). Samples left untreated with MESNA at time 0 represent the total surface Kir2.1 protein. Proteins retaining their biotin tags after the MESNA treatment thus represented surface proteins that had become internalized but not yet recycled back to the plasma membrane. (A) Amounts of Kir2.1 remaining internalized after 0, 5, 10, and 20 min were normalized to total surface expression. Values shown are the average of four separate sets of Western blots. (B) A representative set of Western blots of the Kir2.1 protein internalization assay is shown.