Anti-Cleaved PARP1 antibody [4B5BD2]

• Flow Cyt

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Flow Cytometry - Anti-Cleaved PARP1 antibody [4B5BD2] (AB110315)

Flow cytometry analysis of apoptosis using ab110315. HL-60 cells were treated with 1 µM Staurosporin for 4 hours (blue) or vehicle control (red). Control cells were also stained with an equal amount of an isotype control antibody (black).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cleaved PARP1 antibody [4B5BD2] (AB110315)

Immunocytochemistry images of stained untreated (A) and 4 hours 1 μM Staurosporine-treated (B) Human HeLa cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with 1.0 μg/ml ab110315 for 2 hours at room temperature or over night at 4°C. 10% goat serum was used as the blocking agent for all blocking steps. The secondary antibody was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (in green) used at 2.0 μg/ml for 2 hours. DAPI was used to stain the cell nuclei (in red). Heat induced antigen retrieval (0.1 M Tris-HCl, 5% urea, pH 9.5 for 5 min at 95°C) improves signal. Note that the ab110315 labels only condensed and/or fragmented nuclei of apoptotic Staurosporine-treated cells.

• In-Cell ELISA

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In-Cell ELISA - Anti-Cleaved PARP1 antibody [4B5BD2] (AB110315)

In-Cell ELISA (ICE) using ab110315 on HeLa cells treated with Staurosporine to induce apoptosis. HeLa cells were seeded overnight (50,000 cells/well), treated for 4 hours with 1 µM Staurosporine or with the drug vehicle (DMSO), fixed for Detaching Adherent Cells and analyzed.

• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [4B5BD2] (AB110315)

Lane 1 : Wild type HAP1 (untreated) whole cell lysate (20 μg)
Lane 2 : PARP1 (untreated) knockout HAP1 (untreated) whole cell lysate (20 μg)
Lane 3 : HeLa (untreated) whole cell lysate (20 μg)
Lane 4 : HAP1 (staurosporine treated, 1 uM, 4 hr) whole cell lysate (20 μg)
Lane 5 : PARP1 (staurosporine treated, 1 uM, 4 hr) knockout HAP1 whole cell lysate (20 μg)
Lane 6 : HeLa (staurosporine treated, 1 uM, 4 hr) whole cell lysate (20 μg)

Lanes 1 - 6 : Merged signal (red and green). Green - ab110315 observed at 100 kDa. Red - loading control, ab181602, observed at 37 kDa

ab110315 detected the expected band for cleaved PARP1 in wild type HAP1 cells treated with staurosporine and the band was not seen in PARP1 knockout cells treated with staurosporine. Wild-type and PARP1 knockout samples were subjected to SDS-PAGE. ab110315 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cleaved PARP1 antibody [4B5BD2] (ab110315)

Predicted band size: 113 kDa

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• WB

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Western blot - Anti-Cleaved PARP1 antibody [4B5BD2] (AB110315)

Western Blot analysis using ab110315 antibody and 20 μg of untreated (CON) or 4 hours 1 μM Staurosporine-treated (STS) HeLa cells. Blots were incubated with an antibody that recognizes both the full-length PARP1 and its 89 kDa fragment (left panel), or 1.0 μg/mL PARP1 (cleaved) antibody (ab110315) (right panel). Appropriate HRP-conjugated secondary antibodies followed by ECL detection were used. Note that the MS777 antibody recognizes the apoptosis-specific 89 kDa fragment of PARP1 but it does not recognize the full-length PARP1.

Lanes 1 - 2:

Antibody that recognizes full-length PARP1

Lanes 3 - 4:

Western blot - Anti-Cleaved PARP1 antibody [4B5BD2] (ab110315) at 1 µg/mL

Lanes 1 and 3:

untreated HeLa cells at 20 µg

Lanes 2 and 4:

HeLa cells treated with 1 µM Staurosporinefor 4 hours at 20 µg

Predicted band size: 113 kDa

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• WB

CiteAb

Western blot - Anti-Cleaved PARP1 antibody [4B5BD2] (AB110315)

Cleaved PARP1 western blot using anti-Cleaved PARP1 antibody [4B5BD2] ab110315. Publication image and figure legend from Das, R., Schwintzer, L., et al., 2019, J Cell Sci, PubMed 31138677.

ab110315 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab110315 please see the product overview.

OTUD4 is required for correct stress granule formation. (A) Knockdown of OTUD4 decreases stress granule size and increases stress granule number. HeLa cells were transfected with two different siRNA oligonucleotides (oligo5 and oligo7) against OTUD4 or with control siRNA. OTUD4 and TIAR staining after 30 min arsenite treatment (0.5 mM) reveals differences in stress granule formation in the absence of OTUD4. White arrows indicate residual OTUD4 protein after knockdown, resulting in larger stress granules, resembling control siRNA-transfected cells. Scale bar : 10 μm. The experiment was repeated three times. (B) Quantification of the average number of stress granules per cell. Stress granules were scored in at least 200 cells per condition using CellProfiler software and the average number of granules per cell was depicted in a box plot. (C) Quantification of the average stress granule area. Granule area was determined with CellProfiler software as in B, and size distribution is presented as a box plot. (D) Re-introduction of siRNA-resistant OTUD4 rescues defects in stress granule formation. HeLa cells were transfected with FLAG–OTUD4 or its catalytic inactive mutant (C45A) 24 h after OTUD4 knockdown. Arsenite-treated cells (as in A) were stained with anti-OTUD4 and anti-TIAR antibodies. Scale bar : 20 μm. (E,F) Quantification of granule number and area in 55–100 FLAG–OTUD4-expressing cells per condition was performed as above. Wild-type and C45A-mutated OTUD4 reverse defects in granule formation. Cells with exogenous OTUD4 expression above endogenous levels were omitted for the analysis to exclude overexpression artifacts. In B,C,E,F, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 1–99th percentiles and outliers are indicated. *p<0.05, **p<0.01, ***p<0.001 (B,E, quasi-Poissonian regression analysis; C,F, gamma regression analysis). (G) Analysis of changes in core stress granule proteins and apoptotic markers by western blot. HeLa cells were transfected with OTUD4 siRNA or control siRNA with or without arsenite (30 min, 0.5 mM). Western blot shows that G3BP1 and TIAR levels are unaffected by OTUD4 depletion. Depletion of OTUD4 leads to activation (cleavage) of caspase-3, as detected with anti-cleaved caspase-3 (Asp175) antibody and increased amounts of cleaved PARP, both indicating induction of apoptosis upon loss of OTUD4. The experiment was repeated three times. In D–G, oligo 7 was used for knockdown. (H) Knockdown of OTUD4 enhances the sensitivity of SH-SY5Y cells. SH-SY5Y cells were transfected with siRNA against OTUD4 or control siRNA as indicated. Cells were treated with arsenite (30 min, 0.5 mM) 24 h after siRNA transfection or left untreated. Cells were lysed 6 h later and levels of OTUD4, cleaved caspase-3 and actin were analyzed by western blot. OTUD4-knockdown leads to increased caspase activation in response to arsenite treatment. The experiment was performed three times.

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