pcmv β galactosidase plasmid  (Addgene inc)

Journal: PLoS ONE

Article Title: BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF- β Signaling during Oxidative Stress Responses

doi: 10.1371/journal.pone.0007091

Figure Lengend Snippet: (A) Wild type BRCA1 activates the p3TP-Lux promoter. 0.1 µg of p3TP-Lux and 10 ng of pCMV β -galactosidase plasmids were co-transfected with Flag-Smad3 and HA-BRCA1 plasmids into COS-7 cells, as indicated. A pcDNA3 empty vector plasmid was used to adjust total DNA amounts. At 36 hrs after transfection, luciferase and β -galactosidase assays were performed. The luciferase activities were normalized to β -galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. *, P <0.05 versus empty vector only. (B) Wild type BRCA1 enhances TGF- β -mediated p3TP-Lux promoter activity. 0.1 µg of p3TP-Lux and 10 ng of pCMV β -galactosidase plasmids were co-transfected with 0.25 µg of caT β RII plasmid or 0.1 µg of HA-BRCA1 plasmid into COS-7 cells, as indicated. 24 hrs after transfection, the indicated cells were incubated with 2 ng/ml of TGF- β 1 ligand overnight, followed by luciferase and β -galactosidase assays. The luciferase activities were normalized to β -galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. (C) BRCA1 mutants could not activate the p3TP-Lux promoter. 0.1 µg of p3TP-Lux and 10 ng of pCMV β -galactosidase were co-transfected with 0.1 µg of wild type or various mutant forms of HA-BRCA1 into COS-7 cells. 24 hrs after transfection, the cells were treated with or without 2 ng/ml of TGF-β1 overnight, followed by luciferase and β -galactosidase assays. The luciferase activities were normalized to β -galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. WT: wild type BRCA1; M, P, and Y: BRCA1-M1775R, BRCA1-P1749R, and BRCA1-Y1853x, respectively; N-: BRCA1 (1–683aa); C-: BRCA1 (1301–1863aa).

Article Snippet: The cells were treated with 2 ng/ml of TGF- β 1 overnight 24 hours after transfection. pCMV β -galactosidase plasmid (Clontech, Palo Alto, CA, USA) was used for monitoring transfection efficiency.

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Incubation, Mutagenesis

Journal: PLoS ONE

Article Title: BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF- β Signaling during Oxidative Stress Responses

doi: 10.1371/journal.pone.0007091

Figure Lengend Snippet: (A) Wild type BRCA1 and Smad3 cooperate to increase p3TP-Lux promoter activity. 0.1 µg of p3TP-Lux and 10 ng of pCMV β -galactosidase plasmids were co-transfected with 10 ng of Flag-Smad3, 50 ng of HA-Smad4, and 0.1 µg of wild type HA-BRCA1 plasmids into COS-7 cells, as indicated. 36 hrs after transfection, luciferase and β -galactosidase assays were performed. The luciferase activities were normalized to β -galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. *, P <0.05 versus empty vector only. (B) Wild type BRCA1 increases Smad3 protein abundance. 0.15 µg of Flag-Smad3 plasmid was co-transfected with 0.1 µg, 0.25 µg, and 0.5 µg of wild type HA-BRCA1 or 0.1 µg and 0.25 µg of caT β RII into HEK293 cells. 36 hrs after transfection, 10 µg of total cell lysates was subjected to Western blotting with anti-Flag monoclonal antibody. Anti-CSK polyclonal antibody was used to monitor equal loading. Flag-Smad3 expression levels were normalized to CSK and are represented graphically. This is a representative experiment out of 4 experiments. (C) BRCA1 mutants could not increase Smad3 protein abundance. 0.15 µg of Flag-Smad3 was co-transfected with 0.1 µg of wild type or various mutant forms of HA-BRCA1 into HEK293 cells. 10 µg of total cell lysates was subjected to Western blotting with anti-Flag monoclonal antibody. Anti-CSK polyclonal antibody was used to monitor equal loading. Flag-Smad3 expression levels were normalized to CSK and are represented graphically. This is a representative experiment out of 4 experiments.

Article Snippet: The cells were treated with 2 ng/ml of TGF- β 1 overnight 24 hours after transfection. pCMV β -galactosidase plasmid (Clontech, Palo Alto, CA, USA) was used for monitoring transfection efficiency.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Western Blot, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF- β Signaling during Oxidative Stress Responses

doi: 10.1371/journal.pone.0007091

Figure Lengend Snippet: (A) si-BRCA1 decreases Smad3-Smad4-mediated transcriptional activation of p3TP-Lux reporter in MCF7 cells. MCF7 cells were transfected with 5 nM of siRNAs in 24 well plate. After 16–24 hours, the cells were transfected with p3TP-Lux, pCMV-β-galactosidase, Smad3, Smad4, and empty vector plasmids as indicated. The cells were maintained in cell culture medium containing 0.1% FBS overnight, followed by luciferase and β-galactosidase assays. The fold increase of luciferase activity was obtained from triplicate of three independent experiments, after being normalized by β-glactosidase activity. The Excel software was used to calculate the standard deviation. * P <0.05 as compared to si-GFP treatments+Smad3+Smad4. (B) Depletion of BRCA1 decreases TGF-β-induced Smad3-Smad4 interaction in MCF-7 cells. MCF-7 cells were transfected with 5 nM siGL2 or siBRCA1. After overnight, cells were starved for 3 hours, followed by 1 ng/ml of TGF-β1 treatment for 45 min. Total lysates were subjected to immunoprecipitation with anti-Smad4 antibody and Western blotting with anti-Smad3 antibody as indicated. The membrane was re-probed with anti-Smad4 antibody to monitor the levels of Smad4 protein. The levels of Smad3 protein was detected from 20 µg of total lysatesby using anti-Smad3 antibody. This membrane was reprobed with anti-Actin antibody to monitor equal loading.

Article Snippet: The cells were treated with 2 ng/ml of TGF- β 1 overnight 24 hours after transfection. pCMV β -galactosidase plasmid (Clontech, Palo Alto, CA, USA) was used for monitoring transfection efficiency.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay, Software, Standard Deviation, Immunoprecipitation, Western Blot