Journal: Chemotherapy Research and Practice

Article Title: Inhibition of NF- κ B by Dehydroxymethylepoxyquinomicin Suppresses Invasion and Synergistically Potentiates Temozolomide and γ -Radiation Cytotoxicity in Glioblastoma Cells

doi: 10.1155/2013/593020

Figure Lengend Snippet: (a) The analysis of the methylation status of the MGMT gene in GBM cell lines revealed two groups: methylated (U87MG, U343MG-a, and LN319) and hemimethylated (U251, T98G and U138MG); however, only T98G and U138MG express this gene as detected by real time quantitative PCR (*a pool of 5 white matter samples was used as calibrator); (b) treatment with DHMEQ efficiently decreases the expression of MGMT after 24 h. Data represents two independent experiments in duplicate and are expressed as mean ± SEM; (c) comet assay showed that TMZ-induced DNA damage significantly increases in T98G and U138MG cells as a probable consequence of reduced MGMT expression after exposure to DHMEQ. Each value represents the mean derived from at least three individual experiments (mean ± SD).

Article Snippet: Real-time RT-PCR reactions were performed in triplicate in 10 μ L reactions using the inventoried TaqMan probes (Applied Biosystems, Foster City, CA, EUA) for BCL2 (Hs00608023_m1), BCL-XL (Hs00236329_m1), XIAP (Hs01597783_m1), MMP-2 (Hs01548727_m1), MMP-14 (Hs00237119_m1), uPA (Hs01547054_m1), TIMP-2 (Hs00234278_m1), and MGMT (Hs01037698_m1), on the ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA, EUA).

Techniques: Methylation, Real-time Polymerase Chain Reaction, Expressing, Single Cell Gel Electrophoresis, Derivative Assay

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Cox Regression Analysis of Overall Survival and Progression-Free Survival of 102 Primary Glioblastoma MGMT Unmethylated Patients With Substratification by MGMT Values

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Biomarker Discovery

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Kaplan-Meier analysis is used to A, compare the MGMT (1-1.99) vs MGMT (<1) and MGMT (≥2) patients. The MGMT (1-1.99) group’s median OS (25.4 months) falls in between the MGMT (≥2) (38.8 months) and MGMT (<1) (17.3 months) median OS values (Log-rank P = .001). B, PFS showed the same trend, namely the MGMT (1-1.99) group generated a higher median OS of 11.8 months compared to the MGMT (<1) group but lower than the MGMT (≥ 2) group, yielding 11.8 months vs 7.92 months and 18.0 months (Log-rank P < .0001), respectively. MGMT indicates O-6-methylguanine-DNA methyltransferase; mo, months; OS, overall survival; PFS, progression-free survival.

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Generated

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Cox Regression Analysis of Overall Survival and Progression-Free Survival of 165 Primary Glioblastoma Patients With Substratification by MGMT Values

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Biomarker Discovery

Journal: The Journal of Nutrition

Article Title: Fish Oil Contaminated with Persistent Organic Pollutants Induces Colonic Aberrant Crypt Foci Formation and Reduces Antioxidant Enzyme Gene Expression in Rats

doi: 10.3945/jn.117.251082

Figure Lengend Snippet: Colonic gene expression of Ptgs2 (A), Rela (B), Mgmt (C), Ogg1 (D), Sod (E), and Cat (F) in male rats fed the CO, FO, or POP FO diet for 9 wk. Values are means ± SEs (n = 10). Means without a common letter differ, P < 0.05. Cat, catalase; CO, corn oil; FO, fish oil; Mgmt, O6-methylguanine DNA methyltransferase; Ogg1, 8-oxoguanine glycosylase; POP, persistent organic pollutant; Ptgs2, prostaglandin endoperoxide synthase 2; Rela, component of NF-κB; RQ, relative quantification; Sod, superoxide dismutase.

Article Snippet: The mRNA levels of prostaglandin endoperoxide synthase 2 ( Ptgs2 ), component of NF-κB ( Rela ), superoxide dismutase ( Sod ), catalase, O 6 -methylguanine DNA methyltransferase ( Mgmt ), and 8-oxoguanine glycosylase ( Ogg1 ) were analyzed with the use of quantitative real-time PCR (ViiA7; Applied Biosystems).

Techniques: Gene Expression, Quantitative Proteomics

Journal: The Journal of Nutrition

Article Title: Fish Oil Contaminated with Persistent Organic Pollutants Induces Colonic Aberrant Crypt Foci Formation and Reduces Antioxidant Enzyme Gene Expression in Rats

doi: 10.3945/jn.117.251082

Figure Lengend Snippet: Spearman correlations between variables in male rats fed the CO, FO, or POP FO diet for 9 wk 1

Article Snippet: The mRNA levels of prostaglandin endoperoxide synthase 2 ( Ptgs2 ), component of NF-κB ( Rela ), superoxide dismutase ( Sod ), catalase, O 6 -methylguanine DNA methyltransferase ( Mgmt ), and 8-oxoguanine glycosylase ( Ogg1 ) were analyzed with the use of quantitative real-time PCR (ViiA7; Applied Biosystems).

Techniques:

Journal: Molecular Diagnosis & Therapy

Article Title: Prognostic and Predictive Epigenetic Biomarkers in Oncology

doi: 10.1007/s40291-018-0371-7

Figure Lengend Snippet: Methylation: prognostic and predictive biomarkers with diagnostic utility

Article Snippet: There are a number of commercial tests available to evaluate the MGMT methylation level by (1) methylation-specific polymerase chain reaction (PCR): PredictMDx Glioblastoma (MDx Health); (2) real-time PCR: MGMT Methylation Detection Kit (EntroGen); (3) MS-MLPA: SALSA MS-MLPA probe mix ME011 MMR genes (MRC-Holland); and (4) pyrosequencing technology: PyroMark MGMT Kit (Qiagen).

Techniques: Methylation, Diagnostic Assay, Mutagenesis

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: MGMT expression levels in GBM, GSCs and melanoma cell lines. ( A and D ) The methylation-specific PCR (MSP) analyses of the MGMT promoter from GBMs, GSCs, and melanoma cell lines. Note the bands in the unmethylated (U, 93 bp) and methylated (M, 81 bp) lanes for GBM, GSCs, and melanoma cell lines, reflecting the unmethylated/methylated MGMT promoter. Percentages represent the proportion of methylation (M) and unmethylation (U) on the MGMT promoter in each cell line. ( B and E) Transcript levels of MGMT mRNA in GBM, GSCs, and melanoma cell lines were determined by qRT-PCR. ( C and F) MGMT levels in GBM, GSCs, and melanoma cell lines were determined by immunoblot analysis. Data are the mean ± SEM for three independent experiments.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of siMGMT on the radioresponse of MGMT-producing cells. ( A and B ) ACPK1, GBMJ1, A375, and MM415 cells were transfected with or without 25 nM siMGMT for 48 h. MGMT levels were determined by immunoblot analysis. ( C and D ) ACPK1, GBMJ1, A375, and MM415 cells were transfected with 25 nM of si (negative control) and siMGMT before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by siMGMT alone. Data are the mean ± SEM for three independent experiments. * P < 0.05 and ** P < 0.005 by Student’s t-test.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Transfection, Western Blot, Negative Control, Generated

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of lomeguatrib on radioresponse of MGMT-producing cells. ( A and B ) ACPK1, GBMJ1, A375, and MM415 cells were treated with a gradient (25 µM, 50 µM, 100 µM, and 150 µM) of lomeguatrib for the indicated times (24 h and 48 h). MGMT levels were determined by immunoblot analysis. ( C and D ) ACPK1, GBMJ1, A375, and MM415 cells were treated with the designated concentrations of lomeguatrib (ACPK1, A375, and MM415—100 µM and GBMJ1—50 µM) for 16 h before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by lomeguatrib alone. * P < 0.05 and ** P < 0.005 by Student’s t-test.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Western Blot, Generated

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of lomeguatrib on radiation-induced γH2AX foci and mitotic catastrophe in MGMT-producing cells. ( A ) The quantitative assessment of γH2AX foci per cell at 1 h and 24 h after radiation is shown. Foci were counted in at least 50 cells per experiment and representative histograph images obtained from control, lomeguatrib-only, radiation (4 Gy)-only, and lomeguatrib/radiation combination treatment. Data are the mean ± SEM for three independent experiments, and statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.005 (radiation versus lomeguatrib/radiation). ( B ) The quantitative assessment of nuclear fragmentation per cell at 24 h and 72 h after radiation is shown. Nuclear fragmentation (defined as the presence of two or more distinct lobes within a single cell) was evaluated in at least 100 cells per treatment per experiment and representative histograph images obtained from control cells and cells pretreated with lomeguatrib alone, radiation (4 Gy) alone, and combination treatment of lomeguatrib with radiation. Data are the mean ± SEM for three to four independent experiments. Statistical significance was determined by Student’s t-test. *P < 0.05 and ***P < 0.0005 (radiation versus Lomeguatrib + radiation).

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Control

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of MGMT overexpression on the radioresponse in non-MGMT-producing cells. ( A ) MGMT protein expression was assessed via western blot analysis following transfection of 1 µg GFP-MGMT vector in non-MGMT-producing cells (OSU61, NSC11, WM852 and WM266-4) for 48 h. ( B and C ) OSU61, NSC11, WM852 and WM266-4 cells were transfected with 1 µg of GFP-control vector and GFP-MGMT vector before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by GFP- MGMT vector alone. DMF values were calculated at a surviving fraction (Log) of 0.1. Data are the mean ± SEM for three independent experiments. * P < 0.05 by Student’s t-test.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Generated

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: KDM6B regulates PARP-1 activation and MGMT expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis of PAR and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.

Article Snippet: The membrane was blocked with 5% milk and incubated overnight with primary antibodies: anti-Flag antibody (Sigma, F3165), anti-MGMT antibody (Proteintech, 17195-1-AP), anti-PAR antibody (Trevigen, 4336-BPC-100), anti-PARP-1 antibody (Proteintech, 22999-1-AP), anti-γH2AX antibody (Millipore, 05-636), anti-H2AX antibody (Proteintech, 10856-1-AP), anti-phospho-Chk1 (Ser345) antibody (Cell Signaling Technology, 2348), anti-phospho-RPA32 (Ser33) (Fisher, 50-155-7198), anti-RPA70 (Santa Cruz, SC166023), anti-phospho-ATR (Thr1989) antibody (Cell Signaling Technology, 30632S), anti-ATR antibody (Cell Signaling Technology, 13934S), anti-phospho-ATM (Ser1981) (D6H9) (Cell Signaling Technology, 5883S), anti-ATM (Cell Signaling Technology, 92356S), anti-phospho-Chk1 (Ser296) antibody (Cell Signaling Technology, 2349S), anti-Chk1 antibody (Santa Cruz, sc-8408), anti-phospho-Chk2 (Thr68) (C13C1) antibody (Cell Signaling Technology, 2197S), anti-phospho-Chk2 (D9C6) antibody (Cell Signaling Technology, 6334S), anti-PP2A antibody (Proteintech, 10321-1-AP), anti-H3K27me3 (Cell Signaling Technology, 9733S), anti-XRCC1 antibody (Proteintech, 21468-1-AP), anti-MSH2 antibody (25D12) (Santa Cruz, sc-56163), anti-MSH6 antibody (F-1) (Santa Cruz, sc-271979), or anti-actin antibody (Proteintech, 66009-1-AP) at 4°C, followed by donkey anti-mouse or goat anti-rabbit IgG conjugated to HRP for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Mutagenesis, Immunostaining, Methylation, In Vitro, Activity Assay

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: Pharmacological inhibition or deletion of MGMT sensitizes KDM6BKO cells to alkylating agents. ( A ) Immunoblot analysis of MGMT in SC and KDM6B KO2 HeLa cells 6 h after the treatment of MNNG (50 μM, 15 min) and/or BG (200 μM). ( B and C ) Representative cell death images in SC and KDM6B KO2 HeLa cells 72 h after treatment with MNNG and/or BG (B). PI-positive cells are quantified in C (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. ns, not significant. ( D ) Immunoblot analysis of MGMT in SC, KDM6B KO2, MGMT KO, and KDM6B/MGMT DKO HeLa cells 4 h after MNNG treatment. Numbers indicate the signal intensity. ( E and F ) Representative cell images in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment (E). Cell death is quantified in F (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( G, H ) Representative images (G) and quantification (H) of AIF nuclear translocation in SC and KDM6B KO cells in the presence or absence of BG (200 μM, pretreatment 24 h) and PARP inhibitor Olaparib (10 μM, 30 min pretreatment) at 4 h post MNNG (50 μM, 15 min) treatment. Red, AIF staining; Blue, DAPI staining; Purple, overlay of AIF and DAPI in the nuclei (mean ± SEM, n = 5). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. Scale bar, 10 μm.

Article Snippet: The membrane was blocked with 5% milk and incubated overnight with primary antibodies: anti-Flag antibody (Sigma, F3165), anti-MGMT antibody (Proteintech, 17195-1-AP), anti-PAR antibody (Trevigen, 4336-BPC-100), anti-PARP-1 antibody (Proteintech, 22999-1-AP), anti-γH2AX antibody (Millipore, 05-636), anti-H2AX antibody (Proteintech, 10856-1-AP), anti-phospho-Chk1 (Ser345) antibody (Cell Signaling Technology, 2348), anti-phospho-RPA32 (Ser33) (Fisher, 50-155-7198), anti-RPA70 (Santa Cruz, SC166023), anti-phospho-ATR (Thr1989) antibody (Cell Signaling Technology, 30632S), anti-ATR antibody (Cell Signaling Technology, 13934S), anti-phospho-ATM (Ser1981) (D6H9) (Cell Signaling Technology, 5883S), anti-ATM (Cell Signaling Technology, 92356S), anti-phospho-Chk1 (Ser296) antibody (Cell Signaling Technology, 2349S), anti-Chk1 antibody (Santa Cruz, sc-8408), anti-phospho-Chk2 (Thr68) (C13C1) antibody (Cell Signaling Technology, 2197S), anti-phospho-Chk2 (D9C6) antibody (Cell Signaling Technology, 6334S), anti-PP2A antibody (Proteintech, 10321-1-AP), anti-H3K27me3 (Cell Signaling Technology, 9733S), anti-XRCC1 antibody (Proteintech, 21468-1-AP), anti-MSH2 antibody (25D12) (Santa Cruz, sc-56163), anti-MSH6 antibody (F-1) (Santa Cruz, sc-271979), or anti-actin antibody (Proteintech, 66009-1-AP) at 4°C, followed by donkey anti-mouse or goat anti-rabbit IgG conjugated to HRP for 1 h at room temperature.

Techniques: Inhibition, Western Blot, Translocation Assay, Staining

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: MGMT regulates O 6 MeG levels and O 6 MeG-triggered PARP-1 activation. (A and B) Representative images of O 6 MeG staining ( A ) and quantification ( B ) in SC, KDM6B-KO2, MGMT (MT) KO, KDM6B/MGMT DKO cells with or without MGMT inhibitor BG (200 μM) or PARP inhibitor Olaparib (10 μM) at 0 min, 2 h and 6 h after MNNG treatment (25 μM, 15 min). * P < 0.05, *** P < 0.001, **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. Scale bar, 20 μm. ( C ) Immunoblot analysis of PARP-1 activation and γH2AX in SC and MGMT KO HeLa cells treated with vehicle (−) or MNNG (25 μM, 15 min) for indicated time. ( D ) Immunoblot analysis of PARP-1 activation and γH2AX in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells treated with vehicle (−) or MNNG (50 μM, 15 min) for indicated time. Numbers on the blot indicate the relative signal intensity.

Article Snippet: The membrane was blocked with 5% milk and incubated overnight with primary antibodies: anti-Flag antibody (Sigma, F3165), anti-MGMT antibody (Proteintech, 17195-1-AP), anti-PAR antibody (Trevigen, 4336-BPC-100), anti-PARP-1 antibody (Proteintech, 22999-1-AP), anti-γH2AX antibody (Millipore, 05-636), anti-H2AX antibody (Proteintech, 10856-1-AP), anti-phospho-Chk1 (Ser345) antibody (Cell Signaling Technology, 2348), anti-phospho-RPA32 (Ser33) (Fisher, 50-155-7198), anti-RPA70 (Santa Cruz, SC166023), anti-phospho-ATR (Thr1989) antibody (Cell Signaling Technology, 30632S), anti-ATR antibody (Cell Signaling Technology, 13934S), anti-phospho-ATM (Ser1981) (D6H9) (Cell Signaling Technology, 5883S), anti-ATM (Cell Signaling Technology, 92356S), anti-phospho-Chk1 (Ser296) antibody (Cell Signaling Technology, 2349S), anti-Chk1 antibody (Santa Cruz, sc-8408), anti-phospho-Chk2 (Thr68) (C13C1) antibody (Cell Signaling Technology, 2197S), anti-phospho-Chk2 (D9C6) antibody (Cell Signaling Technology, 6334S), anti-PP2A antibody (Proteintech, 10321-1-AP), anti-H3K27me3 (Cell Signaling Technology, 9733S), anti-XRCC1 antibody (Proteintech, 21468-1-AP), anti-MSH2 antibody (25D12) (Santa Cruz, sc-56163), anti-MSH6 antibody (F-1) (Santa Cruz, sc-271979), or anti-actin antibody (Proteintech, 66009-1-AP) at 4°C, followed by donkey anti-mouse or goat anti-rabbit IgG conjugated to HRP for 1 h at room temperature.

Techniques: Activation Assay, Staining, Western Blot