Patient and control fibroblasts were starved for 1 hour in methionine- and cysteine-free medium and pulsed for 20 minutes with fresh starvation medium containing 50 μCi/ml [³⁵S]methionine and [³⁵S]cysteine protein-labeling mix. Cells were chased in medium containing unlabeled methionine and cysteine for 0, 30, or 60 minutes. Proteins secreted to the medium were precipitated and lysed. (A) Radiolabeled secreted proteins in trichloroacetic acid precipitates were subjected to denaturing gel electrophoresis and visualized by phosphorimaging. ACG1A_1 carries the homozygous LBR mutation described in this study, ACG1A_2 bears biallelic mutations of TRIP11; CTL_2 and 3 are matched wild-type controls (n = 2). (B) Ratio of secreted-to-total proteins at the indicated time points. Data represent mean ± SEM of 3 independent experiments (n = 3; one-way ANOVA with Dunnett’s multiple comparisons test). *P ≤ 0.05, **P < 0.01. (C) Immunoblot analyses of LAMP1, and (D) LAMP2 proteins in whole-cell lysates of ACG1A patient and control fibroblasts. Membranes were overexposed to detect weaker signals. Low-molecular-weight intermediate glycosylation products of LAMP1 in ACG1A_1 are marked by a red asterisk. Similar, but faster migrating protein species are visible in ACG1A_3 (red asterisk). Non-glycosylated forms are present at very low steady state levels in CTL_3 and ACG1A_2 cells. As for LAMP1, the mature LAMP2 proteins in ACG1A run at a lower molecular weight range than in controls. An intermediate product is present in ACG1A_1 cells (~60 kDa, red asterisk).