Figure 1.
Dose response of Ad-SMP30-HA in HEK-293A cells and C3A liver cells.
HEK-293A cells (A) and C3A liver cells (B) were treated with the indicated number of viral particles for 72 h and the expression of SMP30-HA was analyzed by western blotting using SMP30 or HA antibodies.
Figure 2.
Schematic diagram showing the gel areas subjected to LC-MS/MS and peptides recovered therefrom.
Figure 3.
Presence of multiple forms of SMP30 in 7 day old normal rat pup.
Rat liver cytosolic extract in increasing amounts (10, 20 and 30 µg total protein marked as 1, 2 and 3) were subjected to western blotting using antibody against SMP30. Extracts of C3A liver cells expressed SMP30-HA was shown for comparison.
Figure 4.
Presence of multiple forms of SMP30 in the liver and diaphragm of mice injected with 2×1011 Ad-SMP30-HA per animal.
The animals were injected with the control virus (Ad-null, A & B) or Ad-SMP30-HA (C & D) via the tail vein at a dose of 2×1011 VP/animal and sacrificed on day 4 post-virus injection. Tissue homogenates were processed by western blotting using anti-HA antibody.
Figure 5.
Subcellular localization of SMP30 forms in HEK-293A cells 72 h following the infection with Ad-SMP30-HA (20 VP/cell).
A, Fractions obtained by centrifugation; B, Fractions derived using NE-PER nuclear and cytoplasmic extraction kit. The amounts of various forms from different fractions shown in A & B represent the actual amounts shown to be present in the total whole cell lysate. Anti-HA antibody was used to probe the western blots.
Figure 6.
Time course expression of SMP30 and its processed forms in HEK-293A cells and C3A cells.
HEK-293A cell (A) and C3A cells were treated with Ad-SMP30-HA (20 VP/cell for HEK-293A cells and 500 VP/cell for C3A liver cells) for varying periods of time, cell extracts were prepared in M-PER buffer and separated into the particulate and cytosolic fractions. Western blotting was carried out using antibody against HA tag.
Figure 7.
Effect of protease inhibitors on the expression and processing of SMP30 in HEK-293A cells.
A, The inhibitors, MGI32 (10 µM), PSI (50 µM), LCys (10 µM), chloroquine (50 µM) and tunicamycin (3 µg/ml) were added along with Ad-SMP30-HA (20 VP/cell) to the cells and incubated for 24 h; B, Effect of proteasomal degradation system on the processing of SMP30 protein expressed in HEK-293A cells in the presence or absence of 10 µM MG132; C, Varying concentrations of calpain inhibitor I (CI, 5, 10, 15 and 20 µM) or MDL28170 (MDL, 5, 10 and 15 µM) were added to the cells along with Ad-SMP30-HA (20 VP/cell) and incubated for 24 h; D, HEK-293A cells were treated with 20 µM caspase inhibitor I [Z-VAD(OMe)-FMK] or 5 µM β-secretase inhibitor (Z-VLL-CHO) or 5 µM γ-secretase inhibitor (Z-LLNle-CHO) or 10 µM MG132 along with Ad-SMP30-HA (20 VP/cell) for 24 h; Equal volume of DMSO served as control. The cells were harvested, lysed with SDS-PAGE buffer containing 5% β-mercaptoethanol and SDS-PAGE followed by western blotting was carried out using anti-HA antibody.