FIGURE 1.

LAPTM5 stability and localization in macrophages. A, LPS induces down-regulation of LAPTM5. RAW264.7 cells were stimulated with 1 μg/ml LPS for the indicated times. The cells were lysed and immunoblotted with antibodies toward LAPTM5, LAMP2, or actin. B, LAPTM5 protein, but not mRNA, is rapidly down-regulated in BMDMs. BMDMs from WT or LAPTM5 knock-out (KO) mice were stimulated with 100 ng/ml LPS, in duplicate. At the indicated time points, total RNA was isolated, reverse transcribed, and analyzed by quantitative PCR (upper graph), or cells were lysed and total lysates were immunoblotted with anti-LAPTM5 and anti-actin antibodies (lower Western blot panels). N.D. indicates that no LAPTM5 transcripts above base line were detected in KO. The data are presented as the means ± S.D. (n = 4). The results are representative of two independent experiments. C, inhibition of lysosomal degradation leads to accumulation of LAPTM5. RAW264.7 cells were treated for 3 h with 5 μm lactacystine, 20 mm NH₄Cl, 500 nm bafilomycin, 500 nm leupeptin, or dimethyl sulfoxide (DMSO) control. Total cell lysates were immunoblotted with antibodies toward LAPTM5, LAMP2, or ubiquitin. Actin is shown as a control for protein loading. D, LAPTM5 is rapidly degraded in macrophages. RAW264.7 cells were treated with 10 μm cycloheximide (CHX). After the indicated time intervals, the cells were lysed, and the levels of LAPTM5 were detected with anti-LAPTM5 antibodies. Actin controls are shown as well. E, LAPTM5 localizes to lysosome-associated compartments under basal conditions, and LPS stimulation targets LAPTM5 to the lysosomes. Confocal fluorescence analysis of unstimulated RAW264.7 cells (top panels) or stimulated with 1 μg/ml LPS for 15 min (bottom panels) immunostained with anti-LAPTM5 and anti-LAMP1 antibodies to label the late endosome/lysosome. Representative confocal images are shown. The merged panels show an overlay of the two channels. Bar, 5 μm. F, quantification of co-localization between LAPTM5 and LAMP1. Quantification of 22 individual cells, randomly selected and unstimulated (UN) or stimulated with 1 μg/ml LPS for 15 min (LPS), as shown in E. The extent of co-localization between LAPTM5 and LAMP1 is indicated as co-localization coefficient (M2). Statistical analysis was performed using Student's t test. The data are expressed as the means ± S.D. **, p < 0.001.