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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)=Rst-Rbt, where Rst is the percentage
of the total cell ß-Gal activity that is released by samples stimulated
with Ca2+ and Ionomycin, and Rbt 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.
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