Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitor (cocktail, Roche, Basel, Switzerland) and then quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Next, 50 μg of each protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose (NC) membranes (Millipore Corp. Bedford, MA, USA). Following blocked with 5% non-fat milk for 1 h at room temperature, the membranes were probed with primary antibodies against CSE1L (ab96755, 1:2000, Abcam, Cambridge, UK), and p53-binding protein (53BP1) (ab36823, 1:20000, Abcam), flap endonuclease 1 (FEN1) (AB109132, 1:5000, Abcam), β-actin (ab8227, 1:5000, Abcam) overnight at 4 °C. Then the membranes were further incubated with horseradish peroxidase conjugated goat-anti-rabbit secondary antibody (ab205178, 1:10000, Abcam) for 1 h at room temperature. At last, protein signals were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
Ab109132
Manufactured by Abcam
Sourced in United Kingdom
Ab109132 is a primary antibody that can be used for immunofluorescence and immunohistochemistry applications. The antibody targets the protein ALDH1A1. No further details about the intended use or performance of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Sc 23921, by Santa Cruz Biotechnology (3 mentions)
γh2ax, by Cell Signaling Technology (2 mentions)
Sc 20022, by Santa Cruz Biotechnology (2 mentions)
Phosstopphosphotase inhibitors, by Roche (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ab81292, by Abcam (2 mentions)
Nc membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab96755, by Abcam (1 mentions)
Ab205178, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab36823, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ht 8 oxo dg elisa kit 2, by R&D Systems (1 mentions)
Ab26343, by Abcam (1 mentions)
Ab1838, by Abcam (1 mentions)
Ab2893, by Abcam (1 mentions)
Dako pt link system, by Agilent Technologies (1 mentions)
Sc 5285, by Santa Cruz Biotechnology (1 mentions)
7 protocols using ab109132
1
Western Blot Analysis of Protein Targets
2
Antibody-Based Assays for DNA Damage
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Antibodies used in this experiment are listed here: HT 8-oxo-dG ELISA Kit II(R&D Systems China Co, Ltd.No.4380–192-K), APE1(ab189474, Abcam), FEN1(ab109132, Abcam), Pol Beta (ab26343, Abcam) and XRCC1(ab1838, Abcam) antibodies, anti-γ-H2AX antibody (ab2893, Abcam).
3
Compound 1 Induces DNA Damage Response
4
Immunohistochemical Analysis of Prognostic Markers
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Tissue microarray blocks were assembled as previously described.13 (link) For IHC, slides were pretreated using the DAKO PT link system (DAKO; Glostrup, Copenhagen, Denmark) and stained in an Autostainer Plus (DAKO; Glostrup, Copenhagen, Denmark) with the following antibodies: anti-FEN1 (1:1,500, ab109132 Abcam, Cambridge, UK), anti-CPT1A (1:1,800, ab128568, Abcam, Cambridge, UK) and anti-WEE1 (1:200, sc-5285, Santa Cruz, Texas, USA). Slides were scanned at X20 magnification using a NanoZoomer 60 (Hamamatsu Photonics, Shizuoka, Japan) and evaluated with HALO® (Indica Labs, New Mexico, USA). Cox proportional hazard (PH) models were used to estimate the hazard ratio (HR) for CPT1A, FEN and WEE1, using date of diagnosis/treatment start as starting point, and date of death of any cause (BLISS and N-MCL2/3) and date of documented relapse or progression (N-MCL2/3) as end-points for OS and TTP, respectively. The PH assumption was tested using the Therneau and Grambsch test of the Schoenfeld residuals. Maxstat14 was used to define cutoffs, log-rank statistics to evaluate differences between survival curves, and Wilcoxon signed-rank test to compare differences between groups.
5
Protein Expression Analysis in Colorectal Cancer Cells
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SW620, DLD1, HCT-116 and HCT-116 chr3 cells were trypsinised, collected and lysed in Cell Panel Lysis Buffer with complete protease and phosphotase inhibitors. Proteins were separated by gel electrophoresis and transferred to nitrocellulose membrane by Western blot. Membranes were probed, at a concentration of 1:1000 unless stated otherwise, for ATM (sc-23921, Santa Cruz Biotechnology), MSH2 (556349, BD Pharmigen), MLH1 (WH0004292M2, Sigma Aldrich), MSH6 (610918, BD Transduction), MRE11A (ab214, Abcam), RAD50 (611010, BD Transduction), NBS1 (NB100-143, Novus Biologicals), FEN1 (ab109132, Abcam) and β-Actin (A5441, Sigma Aldrich).
6
Western Blot Analysis of DNA Damage Proteins
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Proteins were extracted using RIPA lysis buffer containing 1% PMSF and 1% cocktail protease inhibitor. The protein concentration was determined using the BCA assay kit (PC0020, Solarbio, China). The protein samples were separated on a 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was subsequently incubated with primary antibodies, including FTO (1:2000, ab126605, abcam, UK), POLQ (1:1000, SAB1402530, Merk, Germany), CDC6 (1:2000, ab109315, abcam, UK), CDT1 (1:1000, ab202067, abcam, UK), FEN1 (1:2000, ab109132, abcam, UK), NBN (1:2000, ab32074, abcam, UK), and XRCC1 (1:2000, ab134056, abcam, UK). Subsequently, appropriate secondary antibodies (1:10,000, abcam, UK) were applied for 1 h. The protein bands were visualized using a chemiluminescence imaging system (5200, Tanon, China) for image analysis.
7
Compound 1 Induces DNA Damage Response
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Exponentially growing SW620 cells were seeded in 6-well plates and
incubated for 4 days with compound1 at the stated dose. Cells
were subsequently washed, trypsinized and lysed in Cell Panel Lysis Buffer
(5 mM Tris-HCl, 3 mM EDTA, 3 mM EGTA, 50 mM NaF, 2 mM sodium orthovanadate,
0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate,
and 0.5% Triton X-100) supplemented with complete protease and phosSTOP
phosphotase inhibitors (both Roche). Proteins were separated by gel
electrophoresis and transferred to nitrocellulose membrane by Western blot.
Membranes were probed, at a concentration of 1:1000 unless stated otherwise,
for cleaved PARP (#9541, Cell Signaling Technology), γH2AX (#2577,
Cell Signaling Technology; 1:500), GAPDH (#3683, Cell Signaling Technology;
1:5000), FEN1 (ab109132, Abcam), phospho-ATM (Ser1981) (ab81292, Abcam),
PARP (51-6639GR, BD Biosciences), ATM (sc-23921, Santa Cruz Biotechnology)
and FANCD2 (sc-20022, Santa Cruz Biotechnology).
incubated for 4 days with compound
were subsequently washed, trypsinized and lysed in Cell Panel Lysis Buffer
(5 mM Tris-HCl, 3 mM EDTA, 3 mM EGTA, 50 mM NaF, 2 mM sodium orthovanadate,
0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate,
and 0.5% Triton X-100) supplemented with complete protease and phosSTOP
phosphotase inhibitors (both Roche). Proteins were separated by gel
electrophoresis and transferred to nitrocellulose membrane by Western blot.
Membranes were probed, at a concentration of 1:1000 unless stated otherwise,
for cleaved PARP (#9541, Cell Signaling Technology), γH2AX (#2577,
Cell Signaling Technology; 1:500), GAPDH (#3683, Cell Signaling Technology;
1:5000), FEN1 (ab109132, Abcam), phospho-ATM (Ser1981) (ab81292, Abcam),
PARP (51-6639GR, BD Biosciences), ATM (sc-23921, Santa Cruz Biotechnology)
and FANCD2 (sc-20022, Santa Cruz Biotechnology).
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