Anti-APAF1 antibody [EPR21112-102]

• IP

Supplier Data

Immunoprecipitation - Anti-APAF1 antibody [EPR21112-102] (AB234436)

APAF1 was immunoprecipitated from 0.35 mg HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate with ab234436 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab234436 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1 : HEK-293T whole cell lysate 10 μg (Input).
Lane 2 : ab234436 IP in HEK-293T whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab234436 in HEK-293T whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-APAF1 antibody [EPR21112-102] (ab234436)

Predicted band size: 141 kDa,92 kDa

Observed band size: 94 kDa

false

• WB

Lab

Western blot - Anti-APAF1 antibody [EPR21112-102] (AB234436)

Western blot : Anti-APAF1 antibody [EPR21112-102] (ab234436) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab234436 was shown to bind specifically to APAF1. A band was observed at 140 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in APAF1 knockout cell line. To generate this image, wild-type and APAF1 knockout HCT 116 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.

All lanes:

Western blot - Anti-APAF1 antibody [EPR21112-102] (ab234436) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

APAF1 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

APAF1 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

false

• WB

Supplier Data

Western blot - Anti-APAF1 antibody [EPR21112-102] (AB234436)

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-APAF1 antibody [EPR21112-102] (ab234436) at 1/1000 dilution

Lane 1:

Human fetal heart tissue lysate at 20 µg

Lane 2:

Human fetal kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 141 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-APAF1 antibody [EPR21112-102] (AB234436)

ab234436 was shown to specifically react with APAF1 in wild-type HAP1 cells as signal was lost in APAF1 knockout cells. Wild-type and APAF1 knockout samples were subjected to SDS-PAGE. ab234436 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

Lanes 1,2 and 5 were developed using a higher sensitivity ECL substrate.

Exposure times : Lanes 1-2 : 136 secs; Lanes 3-4 : 3 mins; Lane 5 : 136 secs.

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-APAF1 antibody [EPR21112-102] (ab234436) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

APAF1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen), whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 5:

A20 (mouse reticulum sarcoma B lymphocyte), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 141 kDa

false

• WB

Lab

Western blot - Anti-APAF1 antibody [EPR21112-102] (AB234436)

Western blot : Anti-APAF1 antibody [EPR21112-102] ab234436 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 141 kDa in Wild-type A549 cell lysates with no signal observed at this size in APAF1 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-APAF1 antibody [EPR21112-102] (ab234436) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human APAF1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-apaf1-knockout-a549-cell-line-ab300843'>ab300843</a>) at 20 µg

Lane 3:

Wild-type HCT 116 ab288559 at 20 µg

Lane 4:

Western blot - Human APAF1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-apaf1-knockout-hct116-cell-line-ab300842'>ab300842</a>) at 20 µg

Lane 4:

APAF knockout HCT 116 <a href='/en-us/products/cell-lines/human-apaf1-knockout-hct116-cell-line-ab300842'>ab300842</a> at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 125 kDa

Observed band size: 125 kDa

false

• WB

Supplier Data

Western blot - Anti-APAF1 antibody [EPR21112-102] (AB234436)

Lanes 2 was developed using a higher sensitivity ECL substrate.

Exposure times : Lane 1 : 169 secs; Lane 2 : 3 mins.

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-APAF1 antibody [EPR21112-102] (ab234436) at 1/1000 dilution

Lane 1:

Rat spleen tissue lysate at 20 µg

Lane 2:

Rat liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 141 kDa

false

• WB

CiteAb

Western blot - Anti-APAF1 antibody [EPR21112-102] (AB234436)

APAF1 western blot using anti-APAF1 antibody [EPR21112-102] ab234436. Publication image and figure legend from Gao, Y., Wen, P., et al., 2020, Int J Mol Sci, PubMed 32932732.

ab234436 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 ab234436 please see the product overview.

CDC20 attenuates the sensitivity of HCT116 cells to gamma-rays. (A) CDC20 was stably reduced in HCT116 cells by lentiviral infection. The expression levels of CDC20 were determined by immunoblot analysis (sh-1/2/3 stands for three short hairpin RNA segments). (B) The expression levels of γH2AX and Rad51 were detected by Western blot in control and CDC20 knockdown cells at 24 h after 5 Gy gamma-ray irradiation. (C) The expression level of Rad51 was detected in control and CDC20 overexpression cells at 24 h after 5 Gy gamma-ray irradiation. (D) A clongenic assay of HCT116 cells with reduced CDC20 expression and control vector cells was carried out after irradiation with different doses of gamma-rays. (E) HCT116 cells with reduced CDC20 expression and control vector cells were exposed with the indicated doses of gamma-ray irradiation, and cell viability was measured 24 h later. (F,G) Higher expression level of apaf1, cleaved caspase-9, cleaved caspase-7, and cleaved caspase-3 (F) and more robust caspase-3/7 activation (G) were detected in CDC20 knockdown HCT116 cells compared with control vector cells 24 h after 5 Gy gamma-ray irradiation. (H) HCT116 cells were pretreated with different doses of apcin (0–10 μM) for 24 h before 5 Gy gamma-ray irradiation, and cell viability was detected 24 h after irradiation. (I) Cells were preincubated with apcin (25 or 50 μM) for 24 h before 5 Gy gamma-ray irradiation; then, the expression levels of cleaved caspase-7 and cleaved caspase-3 were determined by Western blot. Actin was used as an internal control. Data were pooled from three independent experiments and the results are represented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

false