FIGURE 4.

Changes in LAMP-2A protein expression were not due to increased cathepsin or calpain abundance in melanomas expressing LAMP-2C. DM331 pCMV and DM331 2C myc cells were incubated overnight at 37°C with ±20 μM CQ (A) or 10 μM calpeptin (calp) (B) to inhibit lysosome proteases or calpain activity, respectively. LAMP-2A levels were detected by western blotting, evaluated by densitometry, and normalized to actin protein levels. LAMP-2A levels were calculated relative to DM331 pCMV cells cultured without CQ or calpeptin. (C) Maturation and gene expression of lysosome proteases cathepsin A (CTSA), cathepsin B (CTSB), and cathepsin D (CTSD) was evaluated in cells overexpressing LAMP-2C. Lysates were resolved by SDS-PAGE and probed to detect the precursor (p), intermediate (i), or mature (m) form of cathepsin A, cathepsin B, and cathepsin D. Protein expression was quantified by densitometry and levels were normalized to actin levels. mRNA levels of CTSA, CTSB, and CTSD transcripts were analyzed by qPCR and normalized to 18S expression. Measurements in (A–C) are relative values calculated by setting the results obtained for DM331 pCMV cells equal to one for comparison to DM331 2C myc cells. Data were analyzed by two-way ANOVA. ^∗∗p < 0.01, and ^∗∗∗p < 0.001 (n = 3).