p130 c 20 sc 317 Search Results


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Santa Cruz Biotechnology p130
Identification of components of the DREAM complex binding to the CDE/CHR element in the mouse cyclin B2 promoter
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p130 c 20 sc 317 affinity  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology p130 c 20 sc 317 affinity
Inhibition of MAGE-A11-dependent AR transcriptional activity by p107. A, pCMV5 (p5) or pCMV-AR (25 ng) was expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 25 ng of empty vector (−), CMV-p107, or <t>pcDNA3-p130</t> with or without 50 ng of pSG5-MAGE (M) and 50 ng pSG5-HA-p300 (P). Cells were plated in medium containing 5% charcoal-stripped serum and incubated for 24 h the day after transfection with or without 1 nm DHT. B, LAPC-4 (LA, lane 1) and HeLa (HL, lane 2) cell extracts (80 μg of protein/lane) from cells cultured in serum-free medium for 24 h before harvest were prepared in immunoblot lysis buffer. Lane 3 contains 0.1 μg of protein extract from COS1 cells (C) expressing pSG5-MAGE. The immunoblot was probed using 10 μg/ml MAGE-A11 antibody. C, pCMV-AR (25 ng) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc and 50 ng of pSG5 (−) or pSG5-MAGE with or without 50 ng of pSG5 (−) or 10, 25, or 50 ng of CMV-p107. Cells were incubated with or without 1 nm DHT. D, pCMV5 (25 ng) or 25 ng of pCMV-AR-(1–660) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc with or without 50 ng of pSG5-MAGE and pCMV5 (−), CMV-p107 and pSG5 (−), or pSG5-MAGE and CMV-p107. E, pCMV5 or pCMV-AR-(1–660) (10 ng) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc, 50 ng of pSG5 (−) or pSG5-MAGE, and 50 ng of p5 (−) or 10, 25, or 50 ng of CMV-p107. F, pCMV5 (5 ng) (−), 5 ng of pCMV-AR-(1–660), or 25 ng of pCMV-AR was expressed in LAPC-4 cells with 0.1 μg of PSA-Enh-Luc and 50 ng of pCMV5 (−) or 50 ng of CMV-p107. Cells were incubated with or without 1 nm DHT. In A and C–F, luciferase activity is the mean ± S.D. (error bars) and representative of three experiments.
P130 C 20 Sc 317 Affinity, supplied by Santa Cruz Biotechnology, 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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Identification of components of the DREAM complex binding to the CDE/CHR element in the mouse cyclin B2 promoter

Journal: Nucleic Acids Research

Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

doi: 10.1093/nar/gkr793

Figure Lengend Snippet: Identification of components of the DREAM complex binding to the CDE/CHR element in the mouse cyclin B2 promoter

Article Snippet: For detection of DREAM complex components, the following antibodies were applied: E2F4 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000 dilution), Tfdp1 (Ab6, Thermo Scientific, 1:400 dilution), p130 (C-20, Santa Cruz Biotech., 1:1000 dilution), p107 (C-18, Santa Cruz Biotech., 1:1000 dilution), Lin9, Lin37, Lin52, Lin54 (1:1000 dilution) ( ) and Rbbp4 (ab79416, Abcam, Cambridge, UK, 1:5000 dilution).

Techniques: Binding Assay

Chromatin immunoprecipitation analysis of DREAM proteins binding to ( A ) mouse or ( B ) human cyclin B2 promoters in vivo . NIH3T3 cells were stably transfected with wild-type, CDE, CHR or CDE/CHR mutant promoter constructs. Nuclear extracts of density-arrested cells were prepared and ChIPs were performed with antibodies targeting E2f4, p130 and Lin9. As a negative control, a non-targeting rabbit antibody was used. All signals are given relative to the input.

Journal: Nucleic Acids Research

Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

doi: 10.1093/nar/gkr793

Figure Lengend Snippet: Chromatin immunoprecipitation analysis of DREAM proteins binding to ( A ) mouse or ( B ) human cyclin B2 promoters in vivo . NIH3T3 cells were stably transfected with wild-type, CDE, CHR or CDE/CHR mutant promoter constructs. Nuclear extracts of density-arrested cells were prepared and ChIPs were performed with antibodies targeting E2f4, p130 and Lin9. As a negative control, a non-targeting rabbit antibody was used. All signals are given relative to the input.

Article Snippet: For detection of DREAM complex components, the following antibodies were applied: E2F4 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000 dilution), Tfdp1 (Ab6, Thermo Scientific, 1:400 dilution), p130 (C-20, Santa Cruz Biotech., 1:1000 dilution), p107 (C-18, Santa Cruz Biotech., 1:1000 dilution), Lin9, Lin37, Lin52, Lin54 (1:1000 dilution) ( ) and Rbbp4 (ab79416, Abcam, Cambridge, UK, 1:5000 dilution).

Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Stable Transfection, Transfection, Mutagenesis, Construct, Negative Control

The MMB complex binds and activates the cyclin B2 promoters through the CHR. ( A ) Nuclear extracts of proliferating NIH3T3 cells were employed for DNA affinity purification with biotinylated DNA probes of the human and mouse cyclin B2 promoters. Protein binding to wild-type, CDE, CHR or CDE/CHR mutant promoter fragments was tested by western blot analysis. As a protein binding to all cyclin B2 probes containing CCAAT-boxes, Nfya was detected. As a negative control, a fragment of the Gapdhs promoter was used. ( B ) Binding of the MMB complex to the mouse and human cyclin B2 DNA probes was assayed with DNA affinity purification of proteins derived from F9 cell nuclear extracts followed by western blot. Note that the DREAM complex components E2f4 and p130 do not bind to the probes. ( C ) To determine the effect of B-myb knockdown on the activity of mouse and human cyclin B2 promoters, NIH3T3 cells were transfected with the reporter constructs mouse Ccnb2 short and human CCNB2 short (wild-type or CHR mutant) together with vectors expressing shRNAs targeting B-myb (sh-Bmyb 1, sh-Bmyb 2). A shRNA construct expressing a non-targeting GFP-shRNA served as a negative control (sh-GFP). Luciferase activities were measured 48 h after transfection.

Journal: Nucleic Acids Research

Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

doi: 10.1093/nar/gkr793

Figure Lengend Snippet: The MMB complex binds and activates the cyclin B2 promoters through the CHR. ( A ) Nuclear extracts of proliferating NIH3T3 cells were employed for DNA affinity purification with biotinylated DNA probes of the human and mouse cyclin B2 promoters. Protein binding to wild-type, CDE, CHR or CDE/CHR mutant promoter fragments was tested by western blot analysis. As a protein binding to all cyclin B2 probes containing CCAAT-boxes, Nfya was detected. As a negative control, a fragment of the Gapdhs promoter was used. ( B ) Binding of the MMB complex to the mouse and human cyclin B2 DNA probes was assayed with DNA affinity purification of proteins derived from F9 cell nuclear extracts followed by western blot. Note that the DREAM complex components E2f4 and p130 do not bind to the probes. ( C ) To determine the effect of B-myb knockdown on the activity of mouse and human cyclin B2 promoters, NIH3T3 cells were transfected with the reporter constructs mouse Ccnb2 short and human CCNB2 short (wild-type or CHR mutant) together with vectors expressing shRNAs targeting B-myb (sh-Bmyb 1, sh-Bmyb 2). A shRNA construct expressing a non-targeting GFP-shRNA served as a negative control (sh-GFP). Luciferase activities were measured 48 h after transfection.

Article Snippet: For detection of DREAM complex components, the following antibodies were applied: E2F4 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000 dilution), Tfdp1 (Ab6, Thermo Scientific, 1:400 dilution), p130 (C-20, Santa Cruz Biotech., 1:1000 dilution), p107 (C-18, Santa Cruz Biotech., 1:1000 dilution), Lin9, Lin37, Lin52, Lin54 (1:1000 dilution) ( ) and Rbbp4 (ab79416, Abcam, Cambridge, UK, 1:5000 dilution).

Techniques: Affinity Purification, Protein Binding, Mutagenesis, Western Blot, Negative Control, Binding Assay, Derivative Assay, Knockdown, Activity Assay, Transfection, Construct, Expressing, shRNA, Luciferase

In silico identification of CHR elements in promoters of genes bound by components of the DREAM complex. ( A ) The 792 promoters have been identified to bind the DREAM complex in ChIP-Seq assays . 149 of these genes (18.8%) possess CHR elements that are located in a distance of 200 bp relative to the transcription start site and that are phylogenetically conserved in vertebrates. As a control, a set of 792 randomly selected genes was assayed using the same parameters. In this control group, 60 conserved CHR elements were identified (7.6%). Using the Fisher's exact test, the overlap between DREAM-binding genes and CHR-containing genes appears to be highly significant ( P = 4.8 × 10 −11 ). ( B ) Randomly selected genes from subgroups of identified genes were further tested by chromatin immunoprecipitation. Nuclear extracts from density-arrested NIH3T3 cells were prepared and ChIPs were performed with antibodies targeting E2f4, p130 and Lin9. As a negative control, a non-targeting rabbit antibody was used (IgG). All signals are given relative to the input.

Journal: Nucleic Acids Research

Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

doi: 10.1093/nar/gkr793

Figure Lengend Snippet: In silico identification of CHR elements in promoters of genes bound by components of the DREAM complex. ( A ) The 792 promoters have been identified to bind the DREAM complex in ChIP-Seq assays . 149 of these genes (18.8%) possess CHR elements that are located in a distance of 200 bp relative to the transcription start site and that are phylogenetically conserved in vertebrates. As a control, a set of 792 randomly selected genes was assayed using the same parameters. In this control group, 60 conserved CHR elements were identified (7.6%). Using the Fisher's exact test, the overlap between DREAM-binding genes and CHR-containing genes appears to be highly significant ( P = 4.8 × 10 −11 ). ( B ) Randomly selected genes from subgroups of identified genes were further tested by chromatin immunoprecipitation. Nuclear extracts from density-arrested NIH3T3 cells were prepared and ChIPs were performed with antibodies targeting E2f4, p130 and Lin9. As a negative control, a non-targeting rabbit antibody was used (IgG). All signals are given relative to the input.

Article Snippet: For detection of DREAM complex components, the following antibodies were applied: E2F4 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000 dilution), Tfdp1 (Ab6, Thermo Scientific, 1:400 dilution), p130 (C-20, Santa Cruz Biotech., 1:1000 dilution), p107 (C-18, Santa Cruz Biotech., 1:1000 dilution), Lin9, Lin37, Lin52, Lin54 (1:1000 dilution) ( ) and Rbbp4 (ab79416, Abcam, Cambridge, UK, 1:5000 dilution).

Techniques: In Silico, ChIP-sequencing, Control, Binding Assay, Chromatin Immunoprecipitation, Negative Control

Inhibition of MAGE-A11-dependent AR transcriptional activity by p107. A, pCMV5 (p5) or pCMV-AR (25 ng) was expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 25 ng of empty vector (−), CMV-p107, or pcDNA3-p130 with or without 50 ng of pSG5-MAGE (M) and 50 ng pSG5-HA-p300 (P). Cells were plated in medium containing 5% charcoal-stripped serum and incubated for 24 h the day after transfection with or without 1 nm DHT. B, LAPC-4 (LA, lane 1) and HeLa (HL, lane 2) cell extracts (80 μg of protein/lane) from cells cultured in serum-free medium for 24 h before harvest were prepared in immunoblot lysis buffer. Lane 3 contains 0.1 μg of protein extract from COS1 cells (C) expressing pSG5-MAGE. The immunoblot was probed using 10 μg/ml MAGE-A11 antibody. C, pCMV-AR (25 ng) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc and 50 ng of pSG5 (−) or pSG5-MAGE with or without 50 ng of pSG5 (−) or 10, 25, or 50 ng of CMV-p107. Cells were incubated with or without 1 nm DHT. D, pCMV5 (25 ng) or 25 ng of pCMV-AR-(1–660) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc with or without 50 ng of pSG5-MAGE and pCMV5 (−), CMV-p107 and pSG5 (−), or pSG5-MAGE and CMV-p107. E, pCMV5 or pCMV-AR-(1–660) (10 ng) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc, 50 ng of pSG5 (−) or pSG5-MAGE, and 50 ng of p5 (−) or 10, 25, or 50 ng of CMV-p107. F, pCMV5 (5 ng) (−), 5 ng of pCMV-AR-(1–660), or 25 ng of pCMV-AR was expressed in LAPC-4 cells with 0.1 μg of PSA-Enh-Luc and 50 ng of pCMV5 (−) or 50 ng of CMV-p107. Cells were incubated with or without 1 nm DHT. In A and C–F, luciferase activity is the mean ± S.D. (error bars) and representative of three experiments.

Journal: The Journal of Biological Chemistry

Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

doi: 10.1074/jbc.M113.468579

Figure Lengend Snippet: Inhibition of MAGE-A11-dependent AR transcriptional activity by p107. A, pCMV5 (p5) or pCMV-AR (25 ng) was expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 25 ng of empty vector (−), CMV-p107, or pcDNA3-p130 with or without 50 ng of pSG5-MAGE (M) and 50 ng pSG5-HA-p300 (P). Cells were plated in medium containing 5% charcoal-stripped serum and incubated for 24 h the day after transfection with or without 1 nm DHT. B, LAPC-4 (LA, lane 1) and HeLa (HL, lane 2) cell extracts (80 μg of protein/lane) from cells cultured in serum-free medium for 24 h before harvest were prepared in immunoblot lysis buffer. Lane 3 contains 0.1 μg of protein extract from COS1 cells (C) expressing pSG5-MAGE. The immunoblot was probed using 10 μg/ml MAGE-A11 antibody. C, pCMV-AR (25 ng) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc and 50 ng of pSG5 (−) or pSG5-MAGE with or without 50 ng of pSG5 (−) or 10, 25, or 50 ng of CMV-p107. Cells were incubated with or without 1 nm DHT. D, pCMV5 (25 ng) or 25 ng of pCMV-AR-(1–660) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc with or without 50 ng of pSG5-MAGE and pCMV5 (−), CMV-p107 and pSG5 (−), or pSG5-MAGE and CMV-p107. E, pCMV5 or pCMV-AR-(1–660) (10 ng) was expressed in HeLa cells with 0.1 μg of PSA-Enh-Luc, 50 ng of pSG5 (−) or pSG5-MAGE, and 50 ng of p5 (−) or 10, 25, or 50 ng of CMV-p107. F, pCMV5 (5 ng) (−), 5 ng of pCMV-AR-(1–660), or 25 ng of pCMV-AR was expressed in LAPC-4 cells with 0.1 μg of PSA-Enh-Luc and 50 ng of pCMV5 (−) or 50 ng of CMV-p107. Cells were incubated with or without 1 nm DHT. In A and C–F, luciferase activity is the mean ± S.D. (error bars) and representative of three experiments.

Article Snippet: Immunoblots were probed using the following antibodies from Santa Cruz Biotechnology, Inc. at 1:200 dilution unless indicated otherwise: p107 C-18 (sc-318) affinity-purified rabbit polyclonal antibody to a carboxyl-terminal human p107 peptide, E2F1 KH95 (sc-251) mouse monoclonal antibody to human E2F1 amino acid residues 342–386, E2F1 C-20 (sc-193) rabbit polyclonal antibody to a carboxyl-terminal epitope, E2F4 C-108 (sc-512) rabbit polyclonal antibody to human E2F4 amino acid residues 108–300, p130 C-20 (sc-317) affinity-purified rabbit polyclonal antibody to a human p130 carboxyl-terminal peptide, and DP-1 K-20 (sc-610) rabbit polyclonal antibody.

Techniques: Inhibition, Activity Assay, Plasmid Preparation, Incubation, Transfection, Cell Culture, Western Blot, Lysis, Expressing, Luciferase

MAGE-A11 interacts with p107 and Rb but not p130. pCMV-FLAG (−), pCMV-FLAG-MAGE, or FLAG-MAGE-(112–429) (5 μg) was expressed in COS1 cells with 5 μg of CMV-p107 (A), pCMV-hRb (B), or pcDNA3-p130 (C). Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and harvested in immunoprecipitation lysis buffer without deoxycholate or glycerol. Cell extracts (50 μg of protein/lane, right) and immunoprecipitates (IP, left) were probed using p107, Rb, p130, and MAGE-A11 antibodies. Arrows, p107, Rb, p130, FLAG-MAGE, and IgG.

Journal: The Journal of Biological Chemistry

Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

doi: 10.1074/jbc.M113.468579

Figure Lengend Snippet: MAGE-A11 interacts with p107 and Rb but not p130. pCMV-FLAG (−), pCMV-FLAG-MAGE, or FLAG-MAGE-(112–429) (5 μg) was expressed in COS1 cells with 5 μg of CMV-p107 (A), pCMV-hRb (B), or pcDNA3-p130 (C). Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and harvested in immunoprecipitation lysis buffer without deoxycholate or glycerol. Cell extracts (50 μg of protein/lane, right) and immunoprecipitates (IP, left) were probed using p107, Rb, p130, and MAGE-A11 antibodies. Arrows, p107, Rb, p130, FLAG-MAGE, and IgG.

Article Snippet: Immunoblots were probed using the following antibodies from Santa Cruz Biotechnology, Inc. at 1:200 dilution unless indicated otherwise: p107 C-18 (sc-318) affinity-purified rabbit polyclonal antibody to a carboxyl-terminal human p107 peptide, E2F1 KH95 (sc-251) mouse monoclonal antibody to human E2F1 amino acid residues 342–386, E2F1 C-20 (sc-193) rabbit polyclonal antibody to a carboxyl-terminal epitope, E2F4 C-108 (sc-512) rabbit polyclonal antibody to human E2F4 amino acid residues 108–300, p130 C-20 (sc-317) affinity-purified rabbit polyclonal antibody to a human p130 carboxyl-terminal peptide, and DP-1 K-20 (sc-610) rabbit polyclonal antibody.

Techniques: Incubation, Immunoprecipitation, Lysis

MAGE-A11 interacts with endogenous p107, stabilizes p107, and modulates p107 ubiquitination. A, pCMV5 or CMV-p107 (2 μg) was expressed in COS1 cells in 10-cm dishes with or without 2 μg of pSG5-MAGE. Cells were incubated for 24 h in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts prepared in immunoblot lysis buffer (40 μg of protein/lane) were probed using p107 and MAGE-A11 antibodies. B, CMV-p107 (3 μg), 3 μg of pCMV5 (−), or 3 μg of pcDNA3-p130 was expressed in COS1 cells in 6-cm dishes with or without 1 μg of pSG5-MAGE. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) prepared in immunoblot lysis buffer were probed using p107, p130, and MAGE-A11 antibodies. C, pCMV5 (2 μg) alone (−) or 2 μg of CMV-p107 with or without 100 ng of WT pSG5-HA-MAGE-(2–429), 25 ng of pSG5-HA-MAGE-(112–429), 50 ng of pSG5-HA-MAGE-(112–307), or 100 ng of pSG5-HA-MAGE-(112–298) were expressed in COS1 cells in 10-cm dishes. Cells were incubated for 24 h in serum-free medium. Cell extracts (40 μg of protein/lane) prepared in immunoblot buffer were probed on transblots using p107 and HA antibodies. D, CMV-p107 (2 μg) and/or 2 μg of pSG5-MAGE were expressed in COS1 cells in 10-cm dishes. Cells were incubated for 24 h in serum-free medium. The next day, medium was exchanged, and cells were treated with 5 μm cycloheximide. Cells were harvested at 0, 8, 16, and 24 h in immunoblot lysis buffer. Cell extracts (50 μg of protein/lane) were probed using p107 and MAGE-A11 antibodies. E, pCMV-FLAG (6 μg) or 6 μg of pCMV-FLAG-ubiquitin (FLAG-Ub) was expressed in COS1 cells with or without 1 μg of pSG5-MAGE and/or 3 μg of CMV-p107. Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and immunoprecipitated using FLAG antibody affinity resin. Transblots of immunoprecipitates (IP, top panels) and cell extracts (40 μg of protein/lane, bottom panels) were probed using p107 and MAGE-A11 antibodies.

Journal: The Journal of Biological Chemistry

Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

doi: 10.1074/jbc.M113.468579

Figure Lengend Snippet: MAGE-A11 interacts with endogenous p107, stabilizes p107, and modulates p107 ubiquitination. A, pCMV5 or CMV-p107 (2 μg) was expressed in COS1 cells in 10-cm dishes with or without 2 μg of pSG5-MAGE. Cells were incubated for 24 h in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts prepared in immunoblot lysis buffer (40 μg of protein/lane) were probed using p107 and MAGE-A11 antibodies. B, CMV-p107 (3 μg), 3 μg of pCMV5 (−), or 3 μg of pcDNA3-p130 was expressed in COS1 cells in 6-cm dishes with or without 1 μg of pSG5-MAGE. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) prepared in immunoblot lysis buffer were probed using p107, p130, and MAGE-A11 antibodies. C, pCMV5 (2 μg) alone (−) or 2 μg of CMV-p107 with or without 100 ng of WT pSG5-HA-MAGE-(2–429), 25 ng of pSG5-HA-MAGE-(112–429), 50 ng of pSG5-HA-MAGE-(112–307), or 100 ng of pSG5-HA-MAGE-(112–298) were expressed in COS1 cells in 10-cm dishes. Cells were incubated for 24 h in serum-free medium. Cell extracts (40 μg of protein/lane) prepared in immunoblot buffer were probed on transblots using p107 and HA antibodies. D, CMV-p107 (2 μg) and/or 2 μg of pSG5-MAGE were expressed in COS1 cells in 10-cm dishes. Cells were incubated for 24 h in serum-free medium. The next day, medium was exchanged, and cells were treated with 5 μm cycloheximide. Cells were harvested at 0, 8, 16, and 24 h in immunoblot lysis buffer. Cell extracts (50 μg of protein/lane) were probed using p107 and MAGE-A11 antibodies. E, pCMV-FLAG (6 μg) or 6 μg of pCMV-FLAG-ubiquitin (FLAG-Ub) was expressed in COS1 cells with or without 1 μg of pSG5-MAGE and/or 3 μg of CMV-p107. Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and immunoprecipitated using FLAG antibody affinity resin. Transblots of immunoprecipitates (IP, top panels) and cell extracts (40 μg of protein/lane, bottom panels) were probed using p107 and MAGE-A11 antibodies.

Article Snippet: Immunoblots were probed using the following antibodies from Santa Cruz Biotechnology, Inc. at 1:200 dilution unless indicated otherwise: p107 C-18 (sc-318) affinity-purified rabbit polyclonal antibody to a carboxyl-terminal human p107 peptide, E2F1 KH95 (sc-251) mouse monoclonal antibody to human E2F1 amino acid residues 342–386, E2F1 C-20 (sc-193) rabbit polyclonal antibody to a carboxyl-terminal epitope, E2F4 C-108 (sc-512) rabbit polyclonal antibody to human E2F4 amino acid residues 108–300, p130 C-20 (sc-317) affinity-purified rabbit polyclonal antibody to a human p130 carboxyl-terminal peptide, and DP-1 K-20 (sc-610) rabbit polyclonal antibody.

Techniques: Incubation, Western Blot, Lysis, Immunoprecipitation

p107 inhibits MAGE-A11 activation of E2F1 in HeLa cells. A, pSG5 (25 ng) (−) or 25 ng of pSG5-MAGE was expressed in HeLa cells with 0.1 μg of E2F1-Luc with or without 50 ng of pSG5 (−) or pSG5-HA-p300 and 10 ng of pCMV5 (−) or CMV-p107. B, pSG5 (50 ng) alone (−), 25 ng of pSG5-MAGE (left), or 25 ng of CMX-E1A (right) was expressed in HeLa cells with 0.1 μg of E2F1-Luc with 50 ng of pCMV5 (−) or 10, 25, or 50 ng of CMV-p107. C, pCMV5 (100 ng) (−) or 100 ng of pSG5-MAGE was expressed in HeLa cells with 0.1 μg of E2F1-Luc and 10 ng of pCMV5 (−), CMV-p107, CMV-p107Δ409–826 (DE) or pcDNA3-p130. Luciferase activity is the mean ± S.D. (error bars) representative of three experiments.

Journal: The Journal of Biological Chemistry

Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

doi: 10.1074/jbc.M113.468579

Figure Lengend Snippet: p107 inhibits MAGE-A11 activation of E2F1 in HeLa cells. A, pSG5 (25 ng) (−) or 25 ng of pSG5-MAGE was expressed in HeLa cells with 0.1 μg of E2F1-Luc with or without 50 ng of pSG5 (−) or pSG5-HA-p300 and 10 ng of pCMV5 (−) or CMV-p107. B, pSG5 (50 ng) alone (−), 25 ng of pSG5-MAGE (left), or 25 ng of CMX-E1A (right) was expressed in HeLa cells with 0.1 μg of E2F1-Luc with 50 ng of pCMV5 (−) or 10, 25, or 50 ng of CMV-p107. C, pCMV5 (100 ng) (−) or 100 ng of pSG5-MAGE was expressed in HeLa cells with 0.1 μg of E2F1-Luc and 10 ng of pCMV5 (−), CMV-p107, CMV-p107Δ409–826 (DE) or pcDNA3-p130. Luciferase activity is the mean ± S.D. (error bars) representative of three experiments.

Article Snippet: Immunoblots were probed using the following antibodies from Santa Cruz Biotechnology, Inc. at 1:200 dilution unless indicated otherwise: p107 C-18 (sc-318) affinity-purified rabbit polyclonal antibody to a carboxyl-terminal human p107 peptide, E2F1 KH95 (sc-251) mouse monoclonal antibody to human E2F1 amino acid residues 342–386, E2F1 C-20 (sc-193) rabbit polyclonal antibody to a carboxyl-terminal epitope, E2F4 C-108 (sc-512) rabbit polyclonal antibody to human E2F4 amino acid residues 108–300, p130 C-20 (sc-317) affinity-purified rabbit polyclonal antibody to a human p130 carboxyl-terminal peptide, and DP-1 K-20 (sc-610) rabbit polyclonal antibody.

Techniques: Activation Assay, Luciferase, Activity Assay