- 35-7000 - Invitrogen Antibodies MGMT antibody

Submitted references Cytotoxic synergy between alisertib and carboplatin versus alisertib and irinotecan are inversely dependent on MGMT levels in glioblastoma cells.

Sak M, Zumbar CT, King PD, Li X, Mifsud CS, Usubalieva A, Anderson CD, Chesnick HM, McElroy JP, Chakravarti A, Burton EC, Lehman NL
Journal of neuro-oncology 2019 Jun;143(2):231-240

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Boyd NH, Walker K, Fried J, Hackney JR, McDonald PC, Benavides GA, Spina R, Audia A, Scott SE, Landis CJ, Tran AN, Bevensee MO, Griguer C, Nozell S, Gillespie GY, Nabors B, Bhat KP, Bar EE, Darley-Usmar V, Xu B, Gordon E, Cooper SJ, Dedhar S, Hjelmeland AB
JCI insight 2017 Dec 21;2(24)

Glioma Cells in the Tumor Periphery Have a Stem Cell Phenotype.

Munthe S, Petterson SA, Dahlrot RH, Poulsen FR, Hansen S, Kristensen BW
PloS one 2016;11(5):e0155106

Phase 2 study of concurrent radiotherapy and temozolomide followed by temozolomide and lomustine in the treatment of children with high-grade glioma: a report of the Children's Oncology Group ACNS0423 study.

Jakacki RI, Cohen KJ, Buxton A, Krailo MD, Burger PC, Rosenblum MK, Brat DJ, Hamilton RL, Eckel SP, Zhou T, Lavey RS, Pollack IF
Neuro-oncology 2016 Oct;18(10):1442-50

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells.

Castro GN, Cayado-Gutiérrez N, Zoppino FC, Fanelli MA, Cuello-Carrión FD, Sottile M, Nadin SB, Ciocca DR
Cell stress & chaperones 2015 Mar;20(2):253-65

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung.

Leng S, Wu G, Collins LB, Thomas CL, Tellez CS, Jauregui AR, Picchi MA, Zhang X, Juri DE, Desai D, Amin SG, Crowell RE, Stidley CA, Liu Y, Swenberg JA, Lin Y, Wathelet MG, Gilliland FD, Belinsky SA
Cancer research 2015 Aug 1;75(15):3108-17

Long-term outcome and MGMT as a predictive marker in 24 patients with atypical pituitary adenomas and pituitary carcinomas given treatment with temozolomide.

Bengtsson D, Schrøder HD, Andersen M, Maiter D, Berinder K, Feldt Rasmussen U, Rasmussen ÅK, Johannsson G, Hoybye C, van der Lely AJ, Petersson M, Ragnarsson O, Burman P
The Journal of clinical endocrinology and metabolism 2015 Apr;100(4):1689-98

Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells.

Qiu ZK, Shen D, Chen YS, Yang QY, Guo CC, Feng BH, Chen ZP
Chinese journal of cancer 2014 Feb;33(2):115-22

Hsp27 (HSPB1): a possible surrogate molecular marker for loss of heterozygosity (LOH) of chromosome 1p in oligodendrogliomas but not in astrocytomas.

Castro GN, Cayado-Gutiérrez N, Moncalero VL, Lima P, De Angelis RL, Chávez V, Cuello-Carrión FD, Ciocca DR
Cell stress & chaperones 2012 Nov;17(6):779-90

Phase II trial of temozolomide in patients with relapsed sensitive or refractory small cell lung cancer, with assessment of methylguanine-DNA methyltransferase as a potential biomarker.

Pietanza MC, Kadota K, Huberman K, Sima CS, Fiore JJ, Sumner DK, Travis WD, Heguy A, Ginsberg MS, Holodny AI, Chan TA, Rizvi NA, Azzoli CG, Riely GJ, Kris MG, Krug LM
Clinical cancer research : an official journal of the American Association for Cancer Research 2012 Feb 15;18(4):1138-45

Temozolomide in the treatment of high-grade gliomas in children: a report from the Children's Oncology Group.

Cohen KJ, Pollack IF, Zhou T, Buxton A, Holmes EJ, Burger PC, Brat DJ, Rosenblum MK, Hamilton RL, Lavey RS, Heideman RL
Neuro-oncology 2011 Mar;13(3):317-23

O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction.

Brell M, Ibáñez J, Tortosa A
BMC cancer 2011 Jan 26;11:35

Impact of morphology, MIB-1, p53 and MGMT on outcome in pilocytic astrocytomas.

Horbinski C, Hamilton RL, Lovell C, Burnham J, Pollack IF
Brain pathology (Zurich, Switzerland) 2010 May;20(3):581-8

Mismatch repair deficiency is an uncommon mechanism of alkylator resistance in pediatric malignant gliomas: a report from the Children's Oncology Group.

Pollack IF, Hamilton RL, Sobol RW, Nikiforova MN, Nikiforov YE, Lyons-Weiler MA, LaFramboise WA, Burger PC, Brat DJ, Rosenblum MK, Gilles FH, Yates AJ, Zhou T, Cohen KJ, Finlay JL, Jakacki RI, Children's Oncology Group
Pediatric blood & cancer 2010 Dec 1;55(6):1066-71

Silencing of MGMT expression by promoter hypermethylation in the metaplasia-dysplasia-carcinoma sequence of Barrett's esophagus.

Kuester D, El-Rifai W, Peng D, Ruemmele P, Kroeckel I, Peters B, Moskaluk CA, Stolte M, Mönkemüller K, Meyer F, Schulz HU, Hartmann A, Roessner A, Schneider-Stock R
Cancer letters 2009 Mar 8;275(1):117-26

Prognostic significance of DNA repair proteins MLH1, MSH2 and MGMT expression in non-small-cell lung cancer and precursor lesions.

Cooper WA, Kohonen-Corish MR, Chan C, Kwun SY, McCaughan B, Kennedy C, Sutherland RL, Lee CS
Histopathology 2008 Apr;52(5):613-22

O6-methylguanine-DNA methyltransferase expression strongly correlates with outcome in childhood malignant gliomas: results from the CCG-945 Cohort.

Pollack IF, Hamilton RL, Sobol RW, Burnham J, Yates AJ, Holmes EJ, Zhou T, Finlay JL
Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2006 Jul 20;24(21):3431-7

Promoter hypermethylation of p16INK4a, E-cadherin, O6-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach.

Schildhaus HU, Kröckel I, Lippert H, Malfertheiner P, Roessner A, Schneider-Stock R
International journal of oncology 2005 Jun;26(6):1493-500

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on nuclear enriched extracts (30 µg lysate) of MCF7 (Lane 1) and Jurkat (Lane 2). The blot was probed with Anti-MGMT Mouse Monoclonal Antibody (Product # 35-7000, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 22 kDa band corresponding to MGMT was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of MGMT was achieved by transfecting HeLa cells with MGMT specific validated siRNAs (Silencer® select Product # s8752). Western blot analysis (Fig. 1) was performed using whole cell extracts from the MGMT knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-MGMT Monoclonal Antibody (Product # 35-7000, 1 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. 2). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to MGMT.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of MGMT (MT23.2) was performed using 70 % confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with MGMT (MT23.2) Mouse Monoclonal antibody (Product # 35-7000) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing predominantly nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical staining of breast carcinoma tissue using Ms anti-MGMT (clone MT23.2) (Product # 35-7000).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. O 6 -methylguanine DNA methyltransferase (MGMT) and nuclear factor-kappaB (NF-kappaB) expression in the GSC lines and the parental glioma cell lines. A, reverse transcription-polymerase chain reaction (RT-PCR) shows that the glioma cell lines U251, SKMG-4, SF295, SKMG-1, U87, MGR1, and MGR2 are MGMT-negative, whereas the SF767 cell line is MGMT-positive. However, the GSC lines U251G, SKMG-4G, SF295G, SKMG-1G, and SF767G become MGMT-positive, whereas the U87G, MGR1G, and MGR2G lines remain MGMT-negative. Meanwhile, all GSC lines and their parental glioma cell lines displayed high NF-kappaB expression. B, the Western blotting data are consistent with the RT-PCR results. C, methylation-specific polymerase chain reaction (MSP) shows MGMT promoter methylation in all GSC lines and their parental glioma cell lines except for the SF767 and SF767G lines. In addition, unmethylated MGMT promoters exist in the glioma cell line SF767 and the GSC lines SF767G, U251G, SKMG-4G, SKMG-1G, and SF295G; H 2 O was used as the blank control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. Inhibiting NF-kappaB expression results in the down-regulation of MGMT expression in the GSC line U251G. RT-PCR (upper panel) and Western blotting (lower panel) data show that TMZ treatment slightly down-regulated MGMT expression but did not affect NF-kappaB expression. MG-132 alone or in combination with TMZ down-regulated both NF-kappaB and MGMT expression. T+M, TMZ plus MG-132.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 3 Double immunofluorescence staining of core and periphery of patient gliomas. Two glioblastomas (AA-BE and CA-DE) were stained with Dapi (blue), IDH1 (green) and Ki-67 (red) and MGMT (red). The software-based classifier is shown in the right column. The classifier illustrates tumor cells co-expressing markers of interest in yellow and tumor cells not co-expressing markers of interest in green. The fluorescence stainings were quantified in both core and periphery for Ki-67 (E,G) and MGMT (F,H). Statistical comparison was performed using student's t-test and ANOVA, ** p< 0.01. Scalebar: 200mum.

35-7000

Antibody data