Figure 5: ARHGAP36 targets PKAC for lysosomal degradation.

(a) HEK293T cells were transfected with PKAC-Flag and YFP-ARHGAP36 or YFP-Cherry control as indicated. Before collecting, cells were pretreated for 30 min with epoxomicin (50 nM) or bafilomycin (100 nM), before cycloheximide (CHX; 50 μg ml⁻¹) addition for a further 6 h. Lysates were immunoblotted with the indicated antibodies. Flag-PKAC levels were densitometrically evaluated and normalized to the first lane shown. (b) MDCK cells were transfected with YFP-ARHGAP36 or YFP control for 8 h and, where indicated, were pretreated for 30 min with epoxomycin (100 nM) or bafilomycin (100 nM), before CHX (1 μg ml⁻¹) addition for a further 8 h. Cells were then fixed after a total of 16 h transfection and subjected to immunofluorescence using antibodies against GFP and PKAC. Images were collected by confocal microscopy. Scale bars, 10 μm. Graph shows the percentage of YFP-ARHGAP36-transfected cells with the respective phenotype, 100 cells counted per condition. (c) Confocal micrographs of MDCK cells transfected with Flag-ARHGAP36 together with GFP-HRS, LAMP1-YFP or GFP-Rab5-QL. Cells were fixed and subjected to immunofluorescence using antibodies against GFP, Flag and PKAC. Scale bars, 10 μm. For Rab5-QL, line scan fluorescence intensity profiles are shown on the right. In red: Rab5-QL, in blue: PKAC. (d) Confocal micrographs of U2OS cells transfected with PKAC-YFP and Flag-ARHGAP36. Cells were fixed and subjected to immunofluorescence using antibodies against GFP, Flag, EEA1 and HRS. Scale bars, 10 μm.