FIGURE 6:

Disruption of BORC/ARL8/HOPS causes missorting of lysosomal luminal protein cathepsin D but not transmembrane proteins. (A, B) The indicated cells were transfected with plasmids encoding cathepsin D-RFP and fixed for immunostaining with antibodies to RFP and LAMP2 at 24 h posttransfection. Cells in each group were analyzed (n = 25, 18, 18, and 22) for cathepsin D-LAMP2 colocalization. The distribution of Pearson correlation coefficient was presented as Pearson correlation coefficient. (C-G) Serum-free culture media were collected after 6 h incubation. Cells were recovered in complete media for 2 h and lysed to prepare cell lysates. The culture media and cell lysates from the indicated cells were subjected to immunoblotting. Quantification was from five independent experiments. (H, I) The indicated cells were fixed for immunostaining of endogenous LAMP2 and LAMTOR4; 20–31 cells in each group from three independent experiments were analyzed. LAMP2-LAMTOR4 colocalization was quantified (n = 31, 25, 29, and 25) and presented as Pearson correlation coefficient; p values were determined by Student’s t test. *p < 0.05, **p < 0.01, ****p < 0.0001, n.s., not significant (vs. WT). Scale bars, 5 μm.