Figure 9. Lysosomal acidification is compromised in RagA/B KO MEFs.

(A) Lysosomal pH measurement in live cells. Cells were fed with OG541-conjugated dextran for overnight followed by serum-starvation for 1 hour, and then trypsinized for spectrofluorimetric measurement. Values represent the mean ± SD of data (n=3 per group). *P < 0.05, Wilcoxon rank sum test. (B) Immunoblot analysis of cathepsin D maturation in lysosome fraction. Lysosome fractions from control and RagA/B KO MEFs were analyzed by immunoblotting using the indicated antibodies. The amount of protein loading was normalized with LAMP1 protein level. Arrowhead indicates the matured form of cathepsin D. (C) Sialidase A treatment increases mobility of LAMP2 on SDS-PAGE. Cell lysates were incubated with sialidase A for 3hrs followed by SDS-PAGE and immunoblotting. (D) Cathepsin D maturation in GFP-TFEB^S142A expressing cells. The amounts of matured cathepsin D in lysosomal fraction of GFP-TFEB^S142A expressing cells were compared with control and RagA/B KO MEFs using immunoblotting. The amount of protein loading was normalized with LAMP2 protein level. Arrowhead indicates the matured form of cathepsin D. (E) A compromised lysosomal acidification capacity in RagA/B KO MEFs. Cells were fed with a pH sensitive fluorescent dye (OG541)-conjugated dextran for overnight, and then starved in a serum-free medium for 2 hours. v-ATPase-mediated acidification of lysosome-enriched fraction was measured in the absence or in the presence of bafilomycin A₁ (100nM) in vitro. The amount of lysosome fraction was normalized with LAMP2 protein level. Each point represents the mean ± S.E of three 30-second intervals from three independent measurements. CON, control MEFs; KO, RagA/B KO MEFs; Baf, bafilomycin A₁.