Fig. 8.

Conformation of LAMP1 surface labeling and intracellular labeling of D52. A: CHO-K1 cells were transfected with Wt-D52, S136/A, or S136/E and treated as control or with 2 μM ionomycin for 5 min. Cells were immunolabeled for external LAMP1 as described in Figs. 5 and 6. Following LAMP1 labeling, coverslips were rinsed 3 times with ice-cold PBS followed by fixation or washed an additional 5 times in acid wash buffer containing (in mM) 100 glycine, 20 mg acetate, 50 KCl, pH 2.2, all at 4°C. After being washed with PBS, cells were fixed in 2% formaldehyde, blocked, permeabilized, and immunolabeled for D52 (1:100) by using Alexa Fluor 546-conjugated anti-rabbit IgG (1:500). Note the complete loss of LAMP1 surface label with acid washing. B: cells were surfaced labeled for LAMP1 as described in Figs. 5 and 6. Following 2% formaldehyde fixation, cells were permeabilized or not with Triton X-100 during labeling with D52 antibodies. Note that cell permeabilization is essential to detect D52 immunoreactivity.