mouse monoclonal anti pold1  (Abcam)

 
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    Name:
    Anti DNA Polymerase delta catalytic subunit antibody 607
    Description:

    Catalog Number:
    AB10362
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    Structured Review

    Abcam mouse monoclonal anti pold1
    Expression of <t>POLD1-R689W</t> alters the specificity of spontaneous mutagenesis in HCT116 cells making it resemble the mutational specificity of HCT15. The diagrams show the HPRT1 mutation spectra of HCT116, HCT116 POLD1-R689W and HCT15 cell lines. The number of independent HPRT1 mutants analyzed (n) is indicated for each spectrum. Data for HCT116 POLD1-R689W and HCT116 are from Table 2 , and data for HCT15 is from reference 25 . Asterisks indicate statistically significant differences in the proportion of indels and GC→TA transversions between HCT116 and HCT116 POLD1-R689W spectra (p=0.000005 for indels and p=0.0043 for GC→TA transversions, Fisher’s exact text). No significant differences were observed between HCT116 POLD1-R689W and HCT15 spectra.

    https://www.bioz.com/result/mouse monoclonal anti pold1/product/Abcam
    Average 93 stars, based on 1 article reviews
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    Images

    1) Product Images from "Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells"

    Article Title: Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells

    Journal: Oncogene

    doi: 10.1038/onc.2017.22

    Expression of POLD1-R689W alters the specificity of spontaneous mutagenesis in HCT116 cells making it resemble the mutational specificity of HCT15. The diagrams show the HPRT1 mutation spectra of HCT116, HCT116 POLD1-R689W and HCT15 cell lines. The number of independent HPRT1 mutants analyzed (n) is indicated for each spectrum. Data for HCT116 POLD1-R689W and HCT116 are from Table 2 , and data for HCT15 is from reference 25 . Asterisks indicate statistically significant differences in the proportion of indels and GC→TA transversions between HCT116 and HCT116 POLD1-R689W spectra (p=0.000005 for indels and p=0.0043 for GC→TA transversions, Fisher’s exact text). No significant differences were observed between HCT116 POLD1-R689W and HCT15 spectra.
    Figure Legend Snippet: Expression of POLD1-R689W alters the specificity of spontaneous mutagenesis in HCT116 cells making it resemble the mutational specificity of HCT15. The diagrams show the HPRT1 mutation spectra of HCT116, HCT116 POLD1-R689W and HCT15 cell lines. The number of independent HPRT1 mutants analyzed (n) is indicated for each spectrum. Data for HCT116 POLD1-R689W and HCT116 are from Table 2 , and data for HCT15 is from reference 25 . Asterisks indicate statistically significant differences in the proportion of indels and GC→TA transversions between HCT116 and HCT116 POLD1-R689W spectra (p=0.000005 for indels and p=0.0043 for GC→TA transversions, Fisher’s exact text). No significant differences were observed between HCT116 POLD1-R689W and HCT15 spectra.

    Techniques Used: Expressing, Mutagenesis

    Immunoblots showing p125 (POLD1) levels in clonal cell lines overexpressing POLD1 or POLD1-R689W . The numbers in parenthesis indicate independently derived cell lines. The left and right panels are from separate SDS-PAGE gels.
    Figure Legend Snippet: Immunoblots showing p125 (POLD1) levels in clonal cell lines overexpressing POLD1 or POLD1-R689W . The numbers in parenthesis indicate independently derived cell lines. The left and right panels are from separate SDS-PAGE gels.

    Techniques Used: Western Blot, Derivative Assay, SDS Page

    DNA sequence context of GC→TA transversions in cancer-related genes in the HCT15 cell line matches the mutational specificity of the POLD1-R689W mutator. (A) Expression of POLD1-R689W results in GC→TA transversions at polypurine/polypyrimidine tracts. Genomic sequence context is shown for all GC→TA transversions observed at the HPRT1 gene of the HCT116 POLD1-R689W cells. Each mutated site is shown as a separate entry with the number of mutations at this site in parentheses. The mutated base is underlined. (B) Sequence context of GC→TA transversions found in a set of 26 cancer-related genes in the HCT15 cell line. The set of genes was the same as the one used previously to characterize the mutational specificity of the NCI-60 panel of cell lines 19 and included APC , ARID1A , BRAF , DNMT1 , DNMT3A , DNMT3B , EGFR , EPHA3 , EPHA5 , EPHA7 , FBXW7 , GRIN2A , KRAS , LRP1B , NF2 , NRAS , PBRM1 , PIK3CA , POLE , PTEN , SETD2 , SPTA1 , STAG2 , SYNE1 , TP53 , and TRRAP . Sequence of the G-containing strand is shown. The mutated base is underlined.
    Figure Legend Snippet: DNA sequence context of GC→TA transversions in cancer-related genes in the HCT15 cell line matches the mutational specificity of the POLD1-R689W mutator. (A) Expression of POLD1-R689W results in GC→TA transversions at polypurine/polypyrimidine tracts. Genomic sequence context is shown for all GC→TA transversions observed at the HPRT1 gene of the HCT116 POLD1-R689W cells. Each mutated site is shown as a separate entry with the number of mutations at this site in parentheses. The mutated base is underlined. (B) Sequence context of GC→TA transversions found in a set of 26 cancer-related genes in the HCT15 cell line. The set of genes was the same as the one used previously to characterize the mutational specificity of the NCI-60 panel of cell lines 19 and included APC , ARID1A , BRAF , DNMT1 , DNMT3A , DNMT3B , EGFR , EPHA3 , EPHA5 , EPHA7 , FBXW7 , GRIN2A , KRAS , LRP1B , NF2 , NRAS , PBRM1 , PIK3CA , POLE , PTEN , SETD2 , SPTA1 , STAG2 , SYNE1 , TP53 , and TRRAP . Sequence of the G-containing strand is shown. The mutated base is underlined.

    Techniques Used: Sequencing, Expressing

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    Western Blot:

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    Article Snippet: .. Antibodies Used for Western Blotting The following antibodies were used for western blotting: β-actin-horseradish peroxidase (HRP) (C4, sc-47778; Santa Cruz Biotechnology), CIAO1 (ab83088; Abcam), Flag-M2, clone M2 (F1804; Sigma-Aldrich), MIP18/FAM96B (20108-1-AP; Proteintech), MMS19 (16015-1-AP; Proteintech), c-myc (A00704-100; Genescript), polymerase delta catalytic subunit (ab10362; Abcam), and XPD (ab54676; Abcam). .. Preparation of Cell Extracts and Immunoprecipitation Experiments Whole cell extracts (WCEs) were prepared using 3 packed cell volumes (PCVs) of buffer A containing 50 mM Na2 HPO4 /NaH2 PO4 (pH 7.4), 150 mM NaCl, 10% glycerol, 0.1% NP-40, 0.5 mM EDTA, 1 mM TCEP (Tris[2-carboxyethyl]phosphine), and protease inhibitors.

    other:

    Article Title: Distinct mechanistic responses to replication fork stalling induced by either nucleotide or protein deprivation
    Article Snippet: The following antibodies were used in this study: rabbit anti-53BP1 (Abcam; cat# ab36823), mouse anti-β-actin (Abcam; cat# ab6276), mouse anti-Chk1 (Cell Singling; cat# 2360S), rabbit anti-p-Chk1-S345 (Cell signaling; cat# 2348S), mouse anti-Chk2 (Cell signaling; cat# 3440S), rabbit anti-p-Chk2-T68 (Cell Signaling; cat# 2661S), mouse anti-DNA polymerase δ, catalytic subunit (Abcam; cat# ab10362), mouse anti-DNA polymerase ε (Santa Cruz Biotechnology; cat# sc-56655), mouse anti-γH2AX-S139 (Millipore; cat# 05–636), rabbit anti-MCM7 (Abcam; cat# ab3732), mouse anti-p53 DO-1 (Santa Cruz Biotechnology; cat# sc-126), rat anti-p58 primase 8D3 (Cell Signaling; cat# 4726S), mouse anti-PCNA F-2 (Santa Cruz Biotechnology; cat# sc-25280), rabbit anti-PML (Santa Cruz Biotechnology; cat# sc-5621), mouse anti-PML (Santa Cruz Biotechnology; cat# sc-966), rabbit anti-RAD51 H-92 (Santa Cruz Biotechnology; cat# sc-8349), rabbit anti-RNA polymerase II CTD p-S2 (Abcam; cat# ab5095), rat anti-RPA32 (Cell Signaling; cat# 2208), rabbit anti-RPA70 (Cell Signaling; cat# 2267).

    Article Title: Break-induced telomere synthesis underlies alternative telomere maintenance
    Article Snippet: The following antibodies were used: anti-BrdU (mouse B44, BD 347580; rat BU1/75, AbD Serotec OBT0030G), anti-ATRX (rabbit H-300, Santa Cruz sc-15408), anti-53BP1 (rabbit, Novus NB100-904), anti-γH2AX (mouse JBW301, Millipore 05-636), anti-Flag (mouse M2, Sigma F1804), anti-PML (mouse PG-M3, Santa Cruz sc-966), anti-Rad51 (rabbit H-92, Santa Cruz sc-8349; mouse 14B4, Abcam ab-213), anti-Hop2/PSMC3IP (rabbit, Novus NBP1-92301), anti-POLD3 (mouse 3E2, Abnova H00010714-M01), anti-POLD1 (mouse 607, Abcam ab10362; rabbit, Bethyl A304-005A), anti-POLD2 (rabbit, Bethyl A304-322A), anti-POLD4 (mouse 2B11, Abnova H00057804-M01A), anti-POLE (mouse 93H3A, Pierce MA5-13616; rabbit, Novus NBP1-68470), anti-POLE3 (rabbit, Bethyl A301-245A), anti-POLA1 (rabbit, Bethyl A302-851A), anti-MCM7 (rabbit, Bethyl A302-584A), anti-MCM4 (rabbit, Bethyl A300-193A), anti-MCM5 (rabbit, Abcam ab75975), anti-RFC1 (rabbit, Bethyl A300-320A), anti-PCNA (mouse PC10, CST #2586) anti-ATR (goat N-17, Santa Cruz sc-1887), anti-PRIM1 (rabbit H300, Santa Cruz sc-366482), anti-Rad17 (goat, Bethyl A300-151A), anti-REV3L (rabbit, GeneTex GTX100153), anti-POLH (rabbit, Bethyl A301-231A), anti-REV1 (rabbit H300, Santa Cruz sc-48806) anti-GAPDH (rabbit 14c10, CST #2118), anti-αTubulin (mouse TU-02, Santa Cruz sc-8035).

    Mass Spectrometry:

    Article Title: A reduction of licensed origins reveals strain-specific replication dynamics in mice
    Article Snippet: .. We used Abcam anti-MCM antibodies (ab3159, ab4460, ab4459, ab17967, ab4458 and ab2360 for MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7, respectively), anti-CDC45 (Abcam; ab56476), anti-Pol delta (Abcam; ab10362), anti-pan actin (Thermo Scientific; MS-1295-P1ABX), anti-p21 (BD Pharmingen; 556431), anti-phospho-p53, anti-phospho-RAD17, anti-γH2AX, anti-RPA32, anti-phospho-CHK1 (Cell Signaling; #9284, #3421, #2577, #2208 and #2341 respectively), and anti-RAD51 (Calbiochem, PC130). ..

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    Abcam mouse monoclonal anti α subunit
    Distribution and expression changes of the <t>α-subunit</t> of the calcium-activated potassium channels ( K Ca channels) in the tumor tissues from the different groups (scale bar = 20 um). (A) Distribution and expression of the α-subunit of the K Ca channels by immunohistochemistry. (B) The α-subunit of the K Ca channels protein expression changes by a Western blot analysis in the tumors from the different groups. The quantification of the α-subunit of the K Ca channels expression was performed by scanning the intensity of the densitometry value ( n = 5 for each group; values represent the mean ± standard deviation). (C) The α-subunit of the K Ca channels mRNA expression changes by a qRT-PCR analysis in the tumors from the different groups ( n = 5 for each group; values represent the mean ± standard deviation). * Indicates a significant difference compared with the control group ( p
    Mouse Monoclonal Anti α Subunit, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse monoclonal anti cox 1
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    Mouse Monoclonal Anti Cox 1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam fitc conjugated mouse monoclonal antibody against low affinity nerve growth factor ngf p75 receptor
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    Fitc Conjugated Mouse Monoclonal Antibody Against Low Affinity Nerve Growth Factor Ngf P75 Receptor, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse monoclonal anti mecp2
    Expression of total <t>MeCP2</t> and MeCP2E1 in primary neurons and astrocytes. A ) Expression of total MeCP2 in embryonic primary cortical neurons and astrocytes are detected by immunofluorescence labelling. Cells were labelled with β-III tubulin (β TUB III) and GFAP to mark neurons and astrocytes, respectively. B ) Expression of MeCP2E1 in primary cortical neurons and astrocytes. Cells were labelled with NEUN and GFAP to mark neurons and astrocytes, respectively. Scale bars represent 5 µm. C ) Western blot analysis of MeCP2E1 levels in primary cortical neurons and astrocytes. The graph depicts the quantification of MeCP2E1 in neurons and astrocytes, p
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    Image Search Results


    Distribution and expression changes of the α-subunit of the calcium-activated potassium channels ( K Ca channels) in the tumor tissues from the different groups (scale bar = 20 um). (A) Distribution and expression of the α-subunit of the K Ca channels by immunohistochemistry. (B) The α-subunit of the K Ca channels protein expression changes by a Western blot analysis in the tumors from the different groups. The quantification of the α-subunit of the K Ca channels expression was performed by scanning the intensity of the densitometry value ( n = 5 for each group; values represent the mean ± standard deviation). (C) The α-subunit of the K Ca channels mRNA expression changes by a qRT-PCR analysis in the tumors from the different groups ( n = 5 for each group; values represent the mean ± standard deviation). * Indicates a significant difference compared with the control group ( p

    Journal: Frontiers in Neuroscience

    Article Title: Increasing of Blood-Brain Tumor Barrier Permeability through Transcellular and Paracellular Pathways by Microbubble-Enhanced Diagnostic Ultrasound in a C6 Glioma Model

    doi: 10.3389/fnins.2017.00086

    Figure Lengend Snippet: Distribution and expression changes of the α-subunit of the calcium-activated potassium channels ( K Ca channels) in the tumor tissues from the different groups (scale bar = 20 um). (A) Distribution and expression of the α-subunit of the K Ca channels by immunohistochemistry. (B) The α-subunit of the K Ca channels protein expression changes by a Western blot analysis in the tumors from the different groups. The quantification of the α-subunit of the K Ca channels expression was performed by scanning the intensity of the densitometry value ( n = 5 for each group; values represent the mean ± standard deviation). (C) The α-subunit of the K Ca channels mRNA expression changes by a qRT-PCR analysis in the tumors from the different groups ( n = 5 for each group; values represent the mean ± standard deviation). * Indicates a significant difference compared with the control group ( p

    Article Snippet: Then, the sections were incubated with a rabbit polyclonal anti-JAM-A antibody (Abcam, Cambridge, UK) and a mouse monoclonal anti-α-subunit of K Ca channels antibody (Abcam, Cambridge, UK) at 4°C overnight.

    Techniques: Expressing, Immunohistochemistry, Western Blot, Standard Deviation, Quantitative RT-PCR

    Expression of COX-1 in the aorta and the heart. Expression of COX 1, in thoracic and abdominal aorta and heart from 6 weeks old, 16 weeks old and 1 year old. Each barr represent the mean±S.E.M. n=5. * p≤0.05.

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Participation of COX-1 and COX-2 in the contractile effect of phenylephrine in prepubescent and old rats

    doi: 10.4196/kjpp.2017.21.4.407

    Figure Lengend Snippet: Expression of COX-1 in the aorta and the heart. Expression of COX 1, in thoracic and abdominal aorta and heart from 6 weeks old, 16 weeks old and 1 year old. Each barr represent the mean±S.E.M. n=5. * p≤0.05.

    Article Snippet: Mouse monoclonal anti-COX-1 (Abcam Cat. # 695), rabbit polyclonal anti-COX-2 Cat. # 15191) and anti-β-actin rabbit polyclonal (Abcam Cat. # ab129348) primary antibodies were diluted in TBS-T plus 5% nonfat dry milk.

    Techniques: Expressing

    Transdifferentiation of mesenchymal stem cells (MSC) to Schwann cells and characterization. Bone marrow stem cells (BMSC) post differentiation showing a bipolar, spindle-shaped morphology with 2-3 processes. (A) Confluent differentiated MSC; (B) DAPI staining of Schwann cell-BMSC; (C) immunofluorescence staining of differentiated MSC: anti-p75-FITC staining and (D) anti-S100-Texas red staining. Scale bar 100 µm

    Journal: Iranian Biomedical Journal

    Article Title: Mesenchymal Stem Cells as an Alternative for Schwann Cells in Rat Spinal Cord Injury

    doi: 10.6091/ibj.1121.2013

    Figure Lengend Snippet: Transdifferentiation of mesenchymal stem cells (MSC) to Schwann cells and characterization. Bone marrow stem cells (BMSC) post differentiation showing a bipolar, spindle-shaped morphology with 2-3 processes. (A) Confluent differentiated MSC; (B) DAPI staining of Schwann cell-BMSC; (C) immunofluorescence staining of differentiated MSC: anti-p75-FITC staining and (D) anti-S100-Texas red staining. Scale bar 100 µm

    Article Snippet: After fixation with 4% paraformaldehyde and blocking with goat serum (Sigma-Aldrich, USA), cells were incubated at 4-8°C overnight with FITC conjugated mouse monoclonal antibody against low-affinity nerve growth factor (NGF) p75 receptor (1:500; Abcam, Canada) and rabbit polyclonal antibody against S100 protein (1:500; Abcam, Canada).

    Techniques: Staining, Immunofluorescence

    Expression of total MeCP2 and MeCP2E1 in primary neurons and astrocytes. A ) Expression of total MeCP2 in embryonic primary cortical neurons and astrocytes are detected by immunofluorescence labelling. Cells were labelled with β-III tubulin (β TUB III) and GFAP to mark neurons and astrocytes, respectively. B ) Expression of MeCP2E1 in primary cortical neurons and astrocytes. Cells were labelled with NEUN and GFAP to mark neurons and astrocytes, respectively. Scale bars represent 5 µm. C ) Western blot analysis of MeCP2E1 levels in primary cortical neurons and astrocytes. The graph depicts the quantification of MeCP2E1 in neurons and astrocytes, p

    Journal: PLoS ONE

    Article Title: Novel MeCP2 Isoform-Specific Antibody Reveals the Endogenous MeCP2E1 Expression in Murine Brain, Primary Neurons and Astrocytes

    doi: 10.1371/journal.pone.0049763

    Figure Lengend Snippet: Expression of total MeCP2 and MeCP2E1 in primary neurons and astrocytes. A ) Expression of total MeCP2 in embryonic primary cortical neurons and astrocytes are detected by immunofluorescence labelling. Cells were labelled with β-III tubulin (β TUB III) and GFAP to mark neurons and astrocytes, respectively. B ) Expression of MeCP2E1 in primary cortical neurons and astrocytes. Cells were labelled with NEUN and GFAP to mark neurons and astrocytes, respectively. Scale bars represent 5 µm. C ) Western blot analysis of MeCP2E1 levels in primary cortical neurons and astrocytes. The graph depicts the quantification of MeCP2E1 in neurons and astrocytes, p

    Article Snippet: Antibodies The following antibodies were used in this study: mouse monoclonal anti-MeCP2 (ab50005, WB-1∶1000; IF-1∶200), rabbit polyclonal anti-H3K9me3 (ab8898, IF-1∶200), rabbit polyclonal anti-H4K20me3 (ab9053, IF-1∶200), mouse monoclonal anti-H3K27me3 (ab6002, IF-1∶200), mouse monoclonal anti-H3K9me2 (ab1220, IF-1∶200) [Abcam]; rabbit polyclonal anti-MeCP2 (07–013, WB-1∶1000, IF-1∶200), mouse monoclonal anti-β-Tubulin III (MAB1637, IF-1∶200), mouse monoclonal anti-NEUN (MAB377, IF-1∶200), chicken polyclonal anti-β-Tubulin III (AB9354, IF-1∶200) [Millipore]; mouse monoclonal anti-C-MYC (A21280, WB-1∶1000, IF-1∶200), mouse monoclonal anti-GFAP (A21282, IF-1∶200) [Molecular Probes]; mouse monoclonal anti-ACTIN (A2228, WB-1∶2500) [Sigma Aldrich].

    Techniques: Expressing, Immunofluorescence, Western Blot

    Expression of total MeCP2 and MeCP2E1 in adult murine brain. Expression of total MeCP2 and MeCP2E1, respectively in olfactory bulb (A1, H1), cerebral cortex (B1, I1), striatum (C1, J1), dentate gyrus of hippocampus (D1, K1), thalamus (E1, L1), cerebellum (F1, M1) and brain stem (G1, N1). Higher magnification images for total MeCP2 (A2–G2) and MeCP2E1 (H2–N2) shows their nuclear expression pattern within the various brain regions. Scale bars: A1–G1; H1–N1 = 80 µm, A2–G2; H2–N2 = 20 µm.

    Journal: PLoS ONE

    Article Title: Novel MeCP2 Isoform-Specific Antibody Reveals the Endogenous MeCP2E1 Expression in Murine Brain, Primary Neurons and Astrocytes

    doi: 10.1371/journal.pone.0049763

    Figure Lengend Snippet: Expression of total MeCP2 and MeCP2E1 in adult murine brain. Expression of total MeCP2 and MeCP2E1, respectively in olfactory bulb (A1, H1), cerebral cortex (B1, I1), striatum (C1, J1), dentate gyrus of hippocampus (D1, K1), thalamus (E1, L1), cerebellum (F1, M1) and brain stem (G1, N1). Higher magnification images for total MeCP2 (A2–G2) and MeCP2E1 (H2–N2) shows their nuclear expression pattern within the various brain regions. Scale bars: A1–G1; H1–N1 = 80 µm, A2–G2; H2–N2 = 20 µm.

    Article Snippet: Antibodies The following antibodies were used in this study: mouse monoclonal anti-MeCP2 (ab50005, WB-1∶1000; IF-1∶200), rabbit polyclonal anti-H3K9me3 (ab8898, IF-1∶200), rabbit polyclonal anti-H4K20me3 (ab9053, IF-1∶200), mouse monoclonal anti-H3K27me3 (ab6002, IF-1∶200), mouse monoclonal anti-H3K9me2 (ab1220, IF-1∶200) [Abcam]; rabbit polyclonal anti-MeCP2 (07–013, WB-1∶1000, IF-1∶200), mouse monoclonal anti-β-Tubulin III (MAB1637, IF-1∶200), mouse monoclonal anti-NEUN (MAB377, IF-1∶200), chicken polyclonal anti-β-Tubulin III (AB9354, IF-1∶200) [Millipore]; mouse monoclonal anti-C-MYC (A21280, WB-1∶1000, IF-1∶200), mouse monoclonal anti-GFAP (A21282, IF-1∶200) [Molecular Probes]; mouse monoclonal anti-ACTIN (A2228, WB-1∶2500) [Sigma Aldrich].

    Techniques: Expressing

    Differential expression of total MeCP2 and MeCP2E1 in adult murine brain regions. Quantification of total MeCP2 ( A ) and MeCP2E1 ( B ) in total cell extracts from the wild type Mecp2 tm1.1Bird y/+ mice whole brain (Brain-WT), olfactory bulb, striatum, cerebral cortex, hippocampus, thalamus, brain stem and cerebellum. Mecp2 tm1.1Bird y/− mice whole brain (Brain-Null) was included as a negative control. Equal loading of protein lysates was verified by probing the same membrane with ACTIN (N = 3±SEM).

    Journal: PLoS ONE

    Article Title: Novel MeCP2 Isoform-Specific Antibody Reveals the Endogenous MeCP2E1 Expression in Murine Brain, Primary Neurons and Astrocytes

    doi: 10.1371/journal.pone.0049763

    Figure Lengend Snippet: Differential expression of total MeCP2 and MeCP2E1 in adult murine brain regions. Quantification of total MeCP2 ( A ) and MeCP2E1 ( B ) in total cell extracts from the wild type Mecp2 tm1.1Bird y/+ mice whole brain (Brain-WT), olfactory bulb, striatum, cerebral cortex, hippocampus, thalamus, brain stem and cerebellum. Mecp2 tm1.1Bird y/− mice whole brain (Brain-Null) was included as a negative control. Equal loading of protein lysates was verified by probing the same membrane with ACTIN (N = 3±SEM).

    Article Snippet: Antibodies The following antibodies were used in this study: mouse monoclonal anti-MeCP2 (ab50005, WB-1∶1000; IF-1∶200), rabbit polyclonal anti-H3K9me3 (ab8898, IF-1∶200), rabbit polyclonal anti-H4K20me3 (ab9053, IF-1∶200), mouse monoclonal anti-H3K27me3 (ab6002, IF-1∶200), mouse monoclonal anti-H3K9me2 (ab1220, IF-1∶200) [Abcam]; rabbit polyclonal anti-MeCP2 (07–013, WB-1∶1000, IF-1∶200), mouse monoclonal anti-β-Tubulin III (MAB1637, IF-1∶200), mouse monoclonal anti-NEUN (MAB377, IF-1∶200), chicken polyclonal anti-β-Tubulin III (AB9354, IF-1∶200) [Millipore]; mouse monoclonal anti-C-MYC (A21280, WB-1∶1000, IF-1∶200), mouse monoclonal anti-GFAP (A21282, IF-1∶200) [Molecular Probes]; mouse monoclonal anti-ACTIN (A2228, WB-1∶2500) [Sigma Aldrich].

    Techniques: Expressing, Mouse Assay, Negative Control

    Validation of the newly developed anti-MeCP2E1 antibody. A) Schematics of MeCP2 isoforms with known functional domains. The difference in the initial amino acids of the N-terminus is highlighted. B) Schematics of the previously reported MECP2E1 (Retro-EF1α-E1) and MECP2E2 (Retro-EF1α-E2) retroviral vectors that were used for transfections (C-D) and transductions (E). C) Western blot experiments with Phoenix cell extracts from control non-transfected (NT), MECP2E1 transfected (E1-T), MECP2E2 transfected (E2-T), and MECP2E1 with peptide competition. Anti-MYC labelling was used as a positive control and ACTIN was used as a loading control. D) Western blot experiments with Phoenix cell extracts from non-transfected cells (NT), and MECP2E1 transfected cells (E1-T), probed with the anti-MeCP2E1 antibody after pre-incubation with increasing concentrations of peptide (0%, 0.1%, 1%, and 5%, of peptide as compared to the amount of antibody used). E) Immunofluorescence staining of NIH3T3 cells transduced with MECP2E1 (top row) or MECP2E2 (bottom row), with the anti-MeCP2E1 and an anti-C-MYC antibody are shown. DAPI signals are shown in blue. Note that the signals in both transduced cells are detectable with anti-C-MYC, but only transduced cells with MECP2E1 show positive signals when incubated with the anti-MeCP2E1 antibody. Scale bars represent 10 µm. MBD: methyl binding domain, ID: intervening domain, TRD: transcriptional repression domain, CTD: C-terminal domain.

    Journal: PLoS ONE

    Article Title: Novel MeCP2 Isoform-Specific Antibody Reveals the Endogenous MeCP2E1 Expression in Murine Brain, Primary Neurons and Astrocytes

    doi: 10.1371/journal.pone.0049763

    Figure Lengend Snippet: Validation of the newly developed anti-MeCP2E1 antibody. A) Schematics of MeCP2 isoforms with known functional domains. The difference in the initial amino acids of the N-terminus is highlighted. B) Schematics of the previously reported MECP2E1 (Retro-EF1α-E1) and MECP2E2 (Retro-EF1α-E2) retroviral vectors that were used for transfections (C-D) and transductions (E). C) Western blot experiments with Phoenix cell extracts from control non-transfected (NT), MECP2E1 transfected (E1-T), MECP2E2 transfected (E2-T), and MECP2E1 with peptide competition. Anti-MYC labelling was used as a positive control and ACTIN was used as a loading control. D) Western blot experiments with Phoenix cell extracts from non-transfected cells (NT), and MECP2E1 transfected cells (E1-T), probed with the anti-MeCP2E1 antibody after pre-incubation with increasing concentrations of peptide (0%, 0.1%, 1%, and 5%, of peptide as compared to the amount of antibody used). E) Immunofluorescence staining of NIH3T3 cells transduced with MECP2E1 (top row) or MECP2E2 (bottom row), with the anti-MeCP2E1 and an anti-C-MYC antibody are shown. DAPI signals are shown in blue. Note that the signals in both transduced cells are detectable with anti-C-MYC, but only transduced cells with MECP2E1 show positive signals when incubated with the anti-MeCP2E1 antibody. Scale bars represent 10 µm. MBD: methyl binding domain, ID: intervening domain, TRD: transcriptional repression domain, CTD: C-terminal domain.

    Article Snippet: Antibodies The following antibodies were used in this study: mouse monoclonal anti-MeCP2 (ab50005, WB-1∶1000; IF-1∶200), rabbit polyclonal anti-H3K9me3 (ab8898, IF-1∶200), rabbit polyclonal anti-H4K20me3 (ab9053, IF-1∶200), mouse monoclonal anti-H3K27me3 (ab6002, IF-1∶200), mouse monoclonal anti-H3K9me2 (ab1220, IF-1∶200) [Abcam]; rabbit polyclonal anti-MeCP2 (07–013, WB-1∶1000, IF-1∶200), mouse monoclonal anti-β-Tubulin III (MAB1637, IF-1∶200), mouse monoclonal anti-NEUN (MAB377, IF-1∶200), chicken polyclonal anti-β-Tubulin III (AB9354, IF-1∶200) [Millipore]; mouse monoclonal anti-C-MYC (A21280, WB-1∶1000, IF-1∶200), mouse monoclonal anti-GFAP (A21282, IF-1∶200) [Molecular Probes]; mouse monoclonal anti-ACTIN (A2228, WB-1∶2500) [Sigma Aldrich].

    Techniques: Functional Assay, Transfection, Western Blot, Positive Control, Incubation, Immunofluorescence, Staining, Transduction, Binding Assay

    Anti-MeCP2E1 validation by immunohistochemistry and MeCP2E1 detection in adult mouse hippocampus. A) A tiled image of MeCP2 immunolabelling in the adult mice hippocampus (A1) is shown. A higher magnification of MeCP2 immunolabelling (A2, red) in hippocampus CA1 region of adult mouse is shown, as well as the merged signals with DAPI signals (blue) in the nuclei (A3). As a negative control for A2–A3, the absence of MeCP2 immunolabelling is shown (A4, red) in the hippocampus CA1 region of Mecp2 tm1.1Bird y/− mouse where DAPI (blue) labelling in nuclei is present and it is shown in the merged image (A5). B) A tiled image of MeCP2E1 immunolabelling in the hippocampus (B1) of adult mice brain. Higher magnification of MeCP2E1 immunolabelling (B2, red) in the hippocampus CA1 region of adult mouse brain, and the merge image with DAPI signals (blue) in the nuclei (B3). The absence of MeCP2E1 immunolabelling (B4) in hippocampus CA1 region of adult mouse brain where DAPI signals (blue) in the nuclei is shown in the merged image (B5). C) Confocal images of MeCP2 (red) immunolabelling in adult mice hippocampus in the CA1 region (C1), shown to overlap with DAPI (blue) nuclear labelling (C2). Signal intensity profile analysis of C2 in two white circles (shown by white arrows), shows enriched MeCP2 signals localized at the DAPI-rich nuclear regions of cells within the hippocampus CA1 (C3). D) Confocal images of MeCP2E1 (red) in wild type adult mouse brain in the hippocampus CA1 region (D1), and overlap with DAPI (blue) nuclear labelling (D2). Signal intensity profile analysis indicates enriched MeCP2E1 (D3) localized at the DAPI-rich regions of hippocampus CA1 nuclei. Scale bars: A2–A5, B2–B5 = 20 µm; A1, B1 = 200 µm.

    Journal: PLoS ONE

    Article Title: Novel MeCP2 Isoform-Specific Antibody Reveals the Endogenous MeCP2E1 Expression in Murine Brain, Primary Neurons and Astrocytes

    doi: 10.1371/journal.pone.0049763

    Figure Lengend Snippet: Anti-MeCP2E1 validation by immunohistochemistry and MeCP2E1 detection in adult mouse hippocampus. A) A tiled image of MeCP2 immunolabelling in the adult mice hippocampus (A1) is shown. A higher magnification of MeCP2 immunolabelling (A2, red) in hippocampus CA1 region of adult mouse is shown, as well as the merged signals with DAPI signals (blue) in the nuclei (A3). As a negative control for A2–A3, the absence of MeCP2 immunolabelling is shown (A4, red) in the hippocampus CA1 region of Mecp2 tm1.1Bird y/− mouse where DAPI (blue) labelling in nuclei is present and it is shown in the merged image (A5). B) A tiled image of MeCP2E1 immunolabelling in the hippocampus (B1) of adult mice brain. Higher magnification of MeCP2E1 immunolabelling (B2, red) in the hippocampus CA1 region of adult mouse brain, and the merge image with DAPI signals (blue) in the nuclei (B3). The absence of MeCP2E1 immunolabelling (B4) in hippocampus CA1 region of adult mouse brain where DAPI signals (blue) in the nuclei is shown in the merged image (B5). C) Confocal images of MeCP2 (red) immunolabelling in adult mice hippocampus in the CA1 region (C1), shown to overlap with DAPI (blue) nuclear labelling (C2). Signal intensity profile analysis of C2 in two white circles (shown by white arrows), shows enriched MeCP2 signals localized at the DAPI-rich nuclear regions of cells within the hippocampus CA1 (C3). D) Confocal images of MeCP2E1 (red) in wild type adult mouse brain in the hippocampus CA1 region (D1), and overlap with DAPI (blue) nuclear labelling (D2). Signal intensity profile analysis indicates enriched MeCP2E1 (D3) localized at the DAPI-rich regions of hippocampus CA1 nuclei. Scale bars: A2–A5, B2–B5 = 20 µm; A1, B1 = 200 µm.

    Article Snippet: Antibodies The following antibodies were used in this study: mouse monoclonal anti-MeCP2 (ab50005, WB-1∶1000; IF-1∶200), rabbit polyclonal anti-H3K9me3 (ab8898, IF-1∶200), rabbit polyclonal anti-H4K20me3 (ab9053, IF-1∶200), mouse monoclonal anti-H3K27me3 (ab6002, IF-1∶200), mouse monoclonal anti-H3K9me2 (ab1220, IF-1∶200) [Abcam]; rabbit polyclonal anti-MeCP2 (07–013, WB-1∶1000, IF-1∶200), mouse monoclonal anti-β-Tubulin III (MAB1637, IF-1∶200), mouse monoclonal anti-NEUN (MAB377, IF-1∶200), chicken polyclonal anti-β-Tubulin III (AB9354, IF-1∶200) [Millipore]; mouse monoclonal anti-C-MYC (A21280, WB-1∶1000, IF-1∶200), mouse monoclonal anti-GFAP (A21282, IF-1∶200) [Molecular Probes]; mouse monoclonal anti-ACTIN (A2228, WB-1∶2500) [Sigma Aldrich].

    Techniques: Immunohistochemistry, Mouse Assay, Negative Control