Journal: Molecular and Cellular Biology
Article Title: Regulation of the cfos serum response element by C/EBPbeta
doi: 10.1128/mcb.17.3.1744
Figure Lengend Snippet: FIG. 1. Size fractionation of SRE BP DNA binding activity. (A) Nuclear extract (nuc ex) from NIH 3T3 cells was fractionated on an SDS–10% polyacryl- amide minigel and transferred to an Immobilon-P membrane, and the separated proteins were renatured in an elution solution as described in Materials and Methods. Gel shift assays were performed with 10 ml of each size fraction and 0.5 ng of 32P-labeled SRE DNA in the presence of 100 ng of poly(dI-dC):poly(dI- dC), as described in Materials and Methods. A scale of apparent molecular masses in kilodaltons, based on protein standards run in parallel on the SDS gel, is shown above lanes 1 to 16. In lane 17, approximately 5 mg of unfractionated nuclear extract from BALB/c 3T3 cells was mixed with 0.5 ng of 32P-labeled SRE DNA in the presence of 500 ng of poly(dI-dC):poly(dI-dC) as described in Materials and Methods. SRF and SRE BP complexes are indicated. (B) Gel shift assays were performed with 5 mg of unfractionated nuclear extract as in panel A, lane 17 (lane 1), 5 ml each of the 38- and 20-kDa SDS-sized fractions mixed at 378C for 20 min prior to DNA addition (lane 2), 5 ml of the 20-kDa SDS-sized fraction (lane 3), or 5 ml of the 38-kDa SDS-sized fraction (lane 4). Protein samples were mixed with 0.5 ng of 32P-labeled SRE DNA in the presence of 100 ng of poly(dI-dC):poly(dI-dC) as described in Materials and Methods except for lane 1, which contained 500 ng of poly(dI-dC):poly(dI-dC). Ten nanograms of aprotinin was added to the protein sample shown in lane 2 prior to incubation at 378C. This figure was prepared with a Mirror 800 color scanner and a Power Macintosh 8100/80 with Adobe Photoshop 2.5.1 and Adobe Illustrator 5.5.
Article Snippet: Approximately 8 mg of NIH 3T3 cell nuclear extract was mixed with 1 mg of BSA (lane 1), 1 ml of guinea pig preimmune serum (lane 2), 1 ml of guinea pig affinity-purified anti-LAP antibody (lane 3), or 1 ml of C/EBPb (lane 4), C/EBPa (lane 5), or E12 (lane 6) peptide antibodies from Santa Cruz Biotechnology in the presence of 20 ng of aprotinin at 48C for 2 h. Protein samples were then mixed with 1 ng of 32P-labeled SRE DNA in the presence of 2 mg of poly(dI-dC):poly(dI-dC) as described in Materials and Methods for buffer I conditions.
Techniques: Fractionation, Binding Assay, Activity Assay, Membrane, Gel Shift, Labeling, SDS-Gel, Incubation
Santa Cruz Biotechnology is a verified supplier 
![FIG. 7. C/EBPb(lz2) inhibits SRE transcription. NIH 3T3 cells were trans- fected as described in Materials and Methods with 10 mg of cfos SRE CAT reporter gene and either 2.5 mg of CMV-4 vector (1CMV vector), 2.5 mg of CMV-C/EBPb(lz2) [1LAP(lz2)], 0.5 mg of CMV-LIP(p20) (1LIP), 2.5 mg of CMV-LAP (p35) (1LAP), or 2.5 mg each of CMV-C/EBPb(lz2) and CMV- LAP [1LAP, LAP(lz2)]. Total DNA in each transfection was adjusted to 15 mg with pUC19 DNA. Twenty-four hours after transfection, cells were serum de- prived for 36 h and all but the plates harvested at time zero were stimulated with 15% fetal calf serum as described in Materials and Methods. Cells were har- vested at the indicated times, and CAT activity was measured as described in Materials and Methods. Fold induction was calculated as the increase in CAT activity relative to that obtained with the reporter gene plus pUC19 DNA in serum-deprived cells at time 0. Values are the averages of three independent experiments, and error bars show standard deviations.](https://doi-unpaywalled-images-cdn.bioz.com/8743/10__1128_slash_mcb__17__3__1744/10__1128_slash_mcb__17__3__1744____page7_image1.jpg)
