Anti-Cleaved Caspase-3 抗体 [E83-77] (ab32042)

Western blot: Anti-CASP3 antibody [E83-77] (ab32042) staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32042 was shown to bind specifically to CASP3. A band was observed at 16/28 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in CASP3 knockout cell line. To generate this image, wild-type and CASP3 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lane 1: Wild type HAP1 + DMSO for 24 hours, whole cell lysate (20 µg)
Lane 2: Wild type HAP1 + 2uM Staurosporine (ab146588) for 24 hours, whole cell lysate (20 µg)
Lane 3: HAP1 CASP3 KO + DMSO for 24 hours, whole cell lysate (20 µg)
Lane 4: HAP1 CASP3 KO + 2uM Staurosporine (ab146588) for 24 hours, whole cell lysate (20 µg)
Lane 5: HeLa + DMSO for 24 hours, whole cell lysate (20 µg)
Lane 6: HeLa + 2uM Staurosporine (ab146588) for 24 hours, whole cell lysate (20 µg)
Lanes 1 - 6: Merged signal (red and green). Green - ab32042 observed at 17 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab32042 was shown to specifically react with CASP3 (Caspase-3) when CASP3 (Caspase-3) knockout samples were used. HAP1 wild-type and CASP3 (Caspase-3) knockout samples were subjected to SDS-PAGE. Ab32042 and ab130007 (Mouse anti vinculin loading control) were incubated overnight at 4°C at 500 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Cells were grown to confluency prior to treatment.

High-glucose induces apoptosis in human Vascular endothelial cells (VECs).

Apoptotic responses in VEC were analyzed by detection of cleaved-Caspase-3 immunofluorescence using ab32042. Cells were treated with low glucose (LG) or high glucose (HG) for 72 hours before treated with 100 ng/mL bFGF, 1 μM sp600125 (sp), or 1 μM U0126 (U) or 10 μM MnTmPyP for 1 hour. Bar = 100 μm.

Ab32042, at dilution of 1/100, staining HeLa (human epithelial cell line from cervix adenocarcinoma) cells by Immunofluorescence.
Left image: control.
Right image: staurosporine treated.