Expression of wild-type RIα in mammalian cells revealed a punctate pattern that is RI specific. (A) Transient transfection of control GFP empty vector or GFP-tagged RIα in HeLa cells (top) and Prkar1a^−/− MEFs (bottom). (B) Expression of fluorescently tagged R-subunit isoforms in HeLa cells (top) and Prkar1a^−/− MEFs (bottom). (C) Coexpression of control GFP empty vector or GFP-tagged RIα with mCherry-tagged PKAc in Prkar1a^−/− MEFs without treatment (left) and with 20 µM forskolin/200 µM IBMX treatment (right). Labels in the top left corner of each image indicate which protein is observed. (D) Representative frames from Video 1 showing Prkar1a^−/− MEFs transiently transfected with GFP-tagged RIα (green) and mCherry-tagged PKAc (red). 10 µM H89 was added to inhibit PKAc activity (last representative frame). (E) Representative frames from Video 3 showing Prkar1a^−/− MEFs transiently transfected with GFP-tagged RIα (green), mCherry-tagged PKAc (red), and CFP-tagged PKI (blue). (D and E) Baseline represents the cell before any treatment, whereas postwash represents the cell after removal of treatment. 20 µM forskolin/200 µM IBMX was added to activate PKAc. DIC, differential interference contrast. wt, wild type. FSK, forskolin. Bars, 20 µm.