mcm5 polyclonal antibody  (Bethyl)

Journal: Ecotoxicology and environmental safety

Article Title: The influence of phenolic environmental estrogen on the transcriptome of uterine leiomyoma cells: A whole transcriptome profiling-based analysis.

doi: 10.1016/j.ecoenv.2021.111945

Figure Lengend Snippet: Fig. 4. The effects of 10.0 μmol/L BPA, 32.0 μmol/L NP on genes and proteins expression in uterine leiomyoma cells. (a) Hierarchically clustered heatmap of differentially expressed genes associated with cell cycle after BPA and NP exposure. (b) E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression by q-PCR presented as relative changes. There were increased E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression in uterine leiomyoma cells treated with10.0 μmol/L BPA, 32.0 μmol/L NP for 48 h compared to control group (*P < 0.05). (c) Representative western blots and (d) band intensity bar graphs of proteins. There were significantly (*P < 0.05) higher expression levels of E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 in the cells treated with BPA and NP for 48 h compared to controls. The western band images shown are representative of three independent experiments and the data were expressed as mean ± SE done in three independent experiments.

Article Snippet: The following primary antibodies were used for the western blotting: E2F1 Monoclonal Antibody (66515-1-Ig; Proteintech; diluted 1:1000); Cyclin D1 Monoclonal Antibody (60186-1-Ig; Proteintech; diluted 1:5000); Cyclin E2 Polyclonal Antibody (11935-1-AP; Proteintech; diluted 1:500); MCM2 Polyclonal Antibody (10513-1-AP; Proteintech; diluted 1:1500); MCM3 Polyclonal Antibody (15597-1-AP; Proteintech; diluted 1:1000); MCM4 Polyclonal Antibody (13043-1-AP; Proteintech; diluted 1:600); MCM5 Polyclonal Antibody (11703-1-AP; Proteintech; diluted 1:1000); MCM6 Polyclonal Antibody (13347-2-AP; Proteintech; diluted 1:8000); phospho-Rb monoclonal (ab184796; abcam; diluted 1:1000); phospho-PI3K polyclonal (ab182651; abcam; diluted 1:500); phospho- AKT polyclonal (ab38449; abcam; diluted 1:500).

Techniques: Expressing, Control, Western Blot

Journal:

Article Title: Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-?

doi: 10.1073/pnas.061487598

Figure Lengend Snippet: GST-Stat1TAD interacts directly with MCM5, but not MCM3. (A) Schematic view of Stat1α and -β and MCM5 and MCM3. The domain structure of Stat1 is according to Chen et al. (7). TAD, transcription activation domain; Y, Tyr-701; S, Ser-727. (B) MCM5 and MCM3 are among the group of Stat1TAD-interacting nuclear proteins. Nuclear extracts from U3A cells were incubated with Sepharose-bead-bound GST or GST-Stat1TAD fusion proteins. The bound proteins were separated by SDS/PAGE and analyzed by Western blotting with indicated antibodies. (C) Stat1TAD interacts directly with MCM5, but not with MCM3. 35S-labeled MCM5 or MCM3 proteins were translated in vitro and incubated with the indicated GST fusion proteins bound to Sepharose beads. The bound proteins were separated by SDS/PAGE and visualized by autoradiography. The bottom panel shows the various GST fusion proteins separated by SDS/PAGE and visualized by Coomassie staining.

Article Snippet: The human MCM5-specific polyclonal antibody was made against bacterially expressed full-length MCM5 proteins (Covance Research Products).

Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Labeling, In Vitro, Autoradiography, Staining

Journal:

Article Title: Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-?

doi: 10.1073/pnas.061487598

Figure Lengend Snippet: MCM5 protein containing mutations of R732 and K734 cannot interact with Stat1TAD. (A) Point mutations in MCM5./indicates a double mutation of residues; KMDA4 is a quintuplet mutation of the ATP-binding site K387 into Met and the conserved DEFD motif (445) into four Ala. Lines on top of the schematic MCM5 molecule indicate previously reported regions of MCM5 that were required for binding to Stat1TAD, with the thickness of the lines representing the strength of interaction. (B) Wild-type or mutant MCM5 proteins were labeled with 35S by in vitro translation and incubated with Sepharose-bead-bound GST or GST-Stat1TAD fusion proteins (GSTS1C). The proteins bound to beads were separated by SDS/PAGE and visualized by autoradiography. Input lanes contain 10% of total input.

Article Snippet: The human MCM5-specific polyclonal antibody was made against bacterially expressed full-length MCM5 proteins (Covance Research Products).

Techniques: Mutagenesis, Binding Assay, Labeling, In Vitro, Incubation, SDS Page, Autoradiography

Journal:

Article Title: Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-?

doi: 10.1073/pnas.061487598

Figure Lengend Snippet: The R732/K734 mutant MCM5 does not interact with Stat1 and other MCM proteins in vivo. (A) Wild-type or mutant MCM5 was tagged at the N terminus with the HA epitope and transiently transfected into 2fTGH cells. Stat1 proteins from whole-cell extracts were precipitated with a Stat1 antibody, and the immune precipitates were separated by SDS/PAGE and analyzed by Western blotting with the indicated antibodies. Input lanes contain 10% of total input. (B) Nuclear extracts from stable cell lines containing the various HA-tagged MCM5 proteins were prepared from cells treated with IFN-γ for 30 min. The HA-tagged MCM5 proteins were precipitated with the anti-HA antibody, and the immune precipitates were separated by SDS/PAGE and analyzed by Western blotting with the indicated antibodies. Input lanes contain 20% of total input.

Article Snippet: The human MCM5-specific polyclonal antibody was made against bacterially expressed full-length MCM5 proteins (Covance Research Products).

Techniques: Mutagenesis, In Vivo, Transfection, SDS Page, Western Blot, Stable Transfection

Journal:

Article Title: Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-?

doi: 10.1073/pnas.061487598

Figure Lengend Snippet: Specific interaction between Stat1 and MCM5 is required for MCM5 to enhance Stat1-mediated transcription activation. (A) Expression plasmids containing HA-tagged wild-type or mutant MCM5 were transiently transfected into U-2 OS cells together with a Stat1-dependent luciferase reporter and the internal control Renilla luciferase reporter (dual luciferase reporter system; Promega). Twenty-four hours after transfection, the cells were either left untreated or treated with IFN-γ for 6 h and harvested for luciferase assays. Results shown are luciferase activities normalized against internal control and the mean + SD of three to five experiments. For MCM5 overexpression samples, only results from treated cells are shown. Vec, the RcCMV plasmid. (B) Western blotting analyses were done with 10 μl of cell lysates used for luciferase assays above. Only lysates from treated cells are shown.

Article Snippet: The human MCM5-specific polyclonal antibody was made against bacterially expressed full-length MCM5 proteins (Covance Research Products).

Techniques: Activation Assay, Expressing, Mutagenesis, Transfection, Luciferase, Over Expression, Plasmid Preparation, Western Blot

Journal:

Article Title: Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-?

doi: 10.1073/pnas.061487598

Figure Lengend Snippet: Coelution of Stat1 and the MCM5/3 subcomplex. Nuclear extracts from IFN-γ-treated (30 min) BUD-8 cells were fractionated by FPLC on a Superose 6 HR10/30 column. 2.5% of total input and 25% of each fraction (fraction numbers are at the top of each lane) were analyzed by Western blotting with the indicated antibodies. Molecular masses were calculated from a calibration curve generated with the Pharmacia HMW calibration kit.

Article Snippet: The human MCM5-specific polyclonal antibody was made against bacterially expressed full-length MCM5 proteins (Covance Research Products).

Techniques: Western Blot, Generated