Figure 2. Disassociated RIα localized to... | Rockefeller University Press

Figure 2.

$Figure 2. Disassociated RIα localized to membranous organelles, and some organelles were ruled out from costaining with organelle markers. (A) Prkar1a+/+ MEFs were treated with 20 µM forskolin (FSK)/200 µM IBMX and fractionated. Cytosol refers to the cytosolic fraction, mem refers to the membrane fraction, nuc refers to the nuclear fraction, and cytosk refers to the cytoskeletal fraction. (B, top) HeLa cells were transiently cotransfected with GFP-tagged RIα (green) and mCherry-tagged LC3 (red). (bottom) Autophagy was induced in Prkar1a−/− MEFs, which were transiently transfected with GFP-tagged RIα (green), and then autophagosomes (LC3) were stained (red). (C) Cells were transfected with GFP-tagged RIα (green). Prkar1a−/− MEFs were stained for lysosomes (LysoTracker), mitochondria (AIF), and peroxisomes (PMP70), whereas HeLa cells were stained for early endosomes (EEA1). (D) Rhodamine-transferrin (Tfn) was used to follow the recycling pathway. (E) Texas red–EGF (EGF) was used to follow the degradative pathway, and cells were fixed at three different time points. EEA1 was stained so that a merge with EGF indicated early endosomes. Bars, 20 µm.$

Disassociated RIα localized to membranous organelles, and some organelles were ruled out from costaining with organelle markers. (A) Prkar1a^+/+ MEFs were treated with 20 µM forskolin (FSK)/200 µM IBMX and fractionated. Cytosol refers to the cytosolic fraction, mem refers to the membrane fraction, nuc refers to the nuclear fraction, and cytosk refers to the cytoskeletal fraction. (B, top) HeLa cells were transiently cotransfected with GFP-tagged RIα (green) and mCherry-tagged LC3 (red). (bottom) Autophagy was induced in Prkar1a^−/− MEFs, which were transiently transfected with GFP-tagged RIα (green), and then autophagosomes (LC3) were stained (red). (C) Cells were transfected with GFP-tagged RIα (green). Prkar1a^−/− MEFs were stained for lysosomes (LysoTracker), mitochondria (AIF), and peroxisomes (PMP70), whereas HeLa cells were stained for early endosomes (EEA1). (D) Rhodamine-transferrin (Tfn) was used to follow the recycling pathway. (E) Texas red–EGF (EGF) was used to follow the degradative pathway, and cells were fixed at three different time points. EEA1 was stained so that a merge with EGF indicated early endosomes. Bars, 20 µm.