Fig. 1. POMC-ß-Gal is processed and secreted in a Ca²⁺-dependent manner. (A) Neuro2A cells expressing either POMC-ß-Gal or ß-Gal were homogenized (H) in homogenization buffer with protease inhibitors and centrifuged at 95,000 rpm for 30 minutes in a Beckman TLA 100.1 to obtain a membrane-containing pellet (P) or cytosol containing supernatant (S) as described previously (Koticha et al., 1999). Equal volumes (40 µl) of the samples were electrophoresed on 9% SDS-PAGE gels, transferred to nitrocellulose membranes and probed with anti-ß-Gal antibodies. The arrowheads indicate bands that are detected with both the antibody against ß-Gal and the antibody against ACTH. The upper arrow indicates the 124 kDa product of POMC-ß-Gal cleavage. The lower arrow indicates ß-Gal. (B) Post-nuclear supernatants (PNS) derived from N2A cells (0.4 ml) were centrifuged at 7,200 g for 10 minutes to obtain a pellet P1 and a supernatant S1. The pellet P1 was re-suspended in 0.4 ml of homogenization buffer. Equal volumes (40 µl) of the fractions were loaded onto an SDS-PAGE gels, transferred to nitrocellulose membranes and probed with anti-Na+/K+ ATPase, anti-ß-Gal antibodies, anti-Mannose-6-Phophate receptor (M6PR) and anti Rab3 (monoclonal 42.1) antibodies. (C) Confocal immunofluorescence of cells stained with anti-Rab3 antibody 42.1. The arrows indicate Rab3 immunoreactivity at the tips. Bar, 10 µm. (D) The supernatant S1 was derived as in B was centrifuged at 70,000 g for 30 minutes, and the pellet was loaded onto a 20-60% (w/v) sucrose density gradient. The gradient was centrifuged at 50,000 rpm in the Beckman TLS-55 swinging bucket rotor. Fractions were collected from the top and analyzed by western blot with anti-mannose-6-phophate receptor antibodies (open triangles), anti-Rab3 antibodies (closed squares) and anti-ß-Gal antibodies (closed circles, the 124/120 kDa band). (E) Neuro2A cells grown in 65 mm wells expressing POMC-ß-Gal were pre-incubated at 37°C for 30 minutes in 1.0 ml M2 buffer with 0.7 mM CaCl₂. Cells were then incubated with 1 ml of M2 with or without 1 µM Ionomycin at 37°C for 1 hour. SDS-PAGE gel lanes were loaded with 50 µl of the cell medium. Western blot analysis of the secreted POMC-ß-Gal products was performed using antibodies against ß-Gal. Densitometry of the bands was done using the NIH Image 1.61 software. This experiment was done three times. (F) Release of ß-Gal activity (% age of total activity in cells) was measured from cells either incubated with Ca²⁺ alone (-) or treated with Ionomycin for 60 minutes (+) or depolarized by KCl for 120 minutes (+), as described in the Materials and Methods. The ß-Gal activity release is an average of data from triplicate samples of a single experiment. This experiment was done four times with similar results. (G) The cells were incubated with Ca²⁺ alone (basal conditions) or with Ca²⁺ and Ionomycin (stimulated conditions) for 0-30, 0-60 and 0-90 minutes. Ca²⁺-dependent ß-Gal release (percentage of total activity in cells)=Rs_t-Rb_t, where Rs_t is the percentage of the total cell ß-Gal activity that is released by samples stimulated with Ca²⁺ and Ionomycin, and Rb_t is the percentage of the total cell ß-Gal activity that is released by samples kept in basal conditions. The average is calculated from the data of three independent experiments done with triplicate samples.