Figure 6.

D52 is present in autophagic vacuoles, and its degradation is blocked by cathepsin B/L inhibition. A, mouse pancreatic acinar cells were pretreated for 30 min with the proteasome inhibitor MG132 (25 μm) or DMSO vehicle (0.1%) before 1 h of supramaximal CCK-8 stimulation (100 nm). Note that D52 levels are significantly reduced by proteasome inhibition alone and the addition of CCK-8 fully depleting cellular D52. B, mouse pancreatic acinar cells were pretreated 30 min with the cathepsin B and L inhibitor Ca-074ME (ME; 50 μm) or DMSO vehicle (0.1%) before 1 h of supramaximal CCK-8 stimulation (100 nm). Note that the depletion of D52 levels by supramaximal CCK-8 is blocked by cathepsin B/L inhibition. C and D, D52 was colocalized with lysosomal membrane protein LAMP2 and LC3 in sections of pancreatic lobules that were treated with maximal (30 pm) or supramaximal (100 nm) CCK-8 for 30 min. Data in D quantifies the number of white puncta representing all three fluorophores under each condition. Note that acinar stimulation rapidly enhances LC3 puncta, which partially colocalizes with D52. Data in A–D are the mean and S.E. (*, p < 0.05) from at least three separate preparations). E, immunoelectron microscopy of D52 in sections of pancreatic lobules treated with 100 pm CCK (2 min). Note that D52 (yellow arrows) is present in autophagic vacuoles (arrowheads) consistent with its colocalization with LAMP2 and LC3 in panel C. A single representative image seen in three independent cell preparations is shown.