Journal: International Journal of Dentistry

Article Title: Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland

doi: 10.1155/2023/1765317

Figure Lengend Snippet: Mouse parotid acinar cells in primary culture. (a) Cell proliferative capacity of BMP2-added (100 ng/mL) and nonadded (control) groups after 48 hr of culture. The proliferative capacity of BMP2-added cultured cells was significantly increased compared to that in the control group. ∗∗ Indicates significance at P < 0.01, ∗ P < 0.05. Data were shown as mean ± SD. Three independent experiments were performed. (b) Protein expression of E-cadherin (an epithelial marker) and vimentin (a mesenchymal marker) in cultured cells of each group at 48 hr after the addition of BMP2. NIH3T3 (3T3) was used as a positive control for mesenchymal markers. RS: rat serum.

Article Snippet: NIH3T3 whole cell lysate (Novus Biologicals, Centennial, CO, USA) was used as a positive control for mesenchymal markers.

Techniques: Control, Cell Culture, Expressing, Marker, Positive Control

Journal: Cell calcium

Article Title: Ca 2+ -dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

doi: 10.1016/j.ceca.2021.102457

Figure Lengend Snippet: Fig. 1. Interaction of S100A6 with cofilin-1 in NIH3T3 fibroblasts. (A) Pull-down assay with the use of protein lysate from NIH3T3 cells and S100A6 affinity resin (upper panel) or empty resin (lower panel). Lanes: 1-input, 2-unbound fraction, 3-last wash, 4-first wash with 0.5 M NaCl, 5-last wash with 0.5 M NaCl, 6-first wash with 1 M NaCl, 7- last wash with 1 M NaCl, 8- elution in buffer containing EGTA. Fractions were analyzed by SDS-PAGE (15% gel) fol lowed by immunoblotting developed with anti- cofilin-1 antibody. (B) Co-immunoprecipitation of S100A6 with cofilin-1 from NIH3T3 cell lysate. 30 μg of protein lysate was used directly for immunoblotting (input; lane 1 in both upper and lower panel) and 2.5 mg of protein lysate was incubated with (upper panel) or without (control, lower panel) anti-S100A6 monoclonal antibody and then with protein A/G agarose. In both panels, lane 2 shows unbound fraction, lane 3-last wash and lane 4-elution. Proteins were identified by immunoblotting using anti- cofilin-1 antibody. (C) Presence of S100A6- cofilin-1 complexes in NIH3T3 cells studied by PLA. Complexes of examined proteins are visualized in red; cell nuclei, stained with DAPI, are in blue. Scale bar is 20 μm.

Article Snippet: For co-immunoprecipitation assays 2.5 mg of protein lysate from NIH3T3 cells, obtained using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer’s instruction was incubated with protein A/G-Agarose (Santa Cruz Biotechnology) for 1 h at 4◦C, as described by Jurewicz et al. [31].

Techniques: Pull Down Assay, SDS Page, Western Blot, Immunoprecipitation, Incubation, Control, Staining

Journal: Molecular Cancer

Article Title: NF-κB/STAT3/PI3K signaling crosstalk in iMyc Eμ B lymphoma

doi: 10.1186/1476-4598-9-97

Figure Lengend Snippet: AKT is constitutively phosphorylated, in a PTEN independent-manner, in a majority of LBLs and iMyc Eμ -1 cells . (A) Western blot analysis of the activating-phosphorylation status of key proteins of the PI3K (AKT, P-AKT S473 and T308), MAPK (ERK 1/2, P-ERK1/2, total p 38, P-p 38) and mTOR (p70S6K, P-p70S6K) signaling pathways. Positive controls for P-ERK1/2, P-p38 and P-p70S6K were from extracts of UV-treated HeLa cells, NIH 3T3 cells and insulin-treated MCF-7 cells, respectively. (B and C) Levels of PTEN protein (B) and mRNA (C) in LBLs and iMyc Eμ -1. α-tubulin and β-actin served as loading controls, respectively. "C" denotes control BL6 splenic B cells.

Article Snippet: Total cell extracts from UV-treated HeLa and NIH 3T3 cells were used as positive controls for P-ERK1/2 (sc-2221) and P-p38 (sc-2210), respectively (Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Protein-Protein interactions, Control

Journal: Cell Death and Differentiation

Article Title: BRCA1 and BRCA2 gene expression: p53- and cell cycle-dependent repression requires RB and DREAM

doi: 10.1038/s41418-025-01566-9

Figure Lengend Snippet: Wild-type (WT), Lin37 -/- (Lin37-KO), Rb -/- (Rb-KO) or Rb -/- /Lin37 -/- (DKO) NIH3T3 cells were used. Brca1 ( A ) and Brca2 ( B ) mRNA expression was measured from proliferating, density-arrested, or serum-starved cells. A , B Mean ± SD; n = 4, two-way ANOVA; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Analogous experiments were performed with serum-starved cells predominantly in G 0 and cells serum-restimulated to re-enter the cell cycle. From G 0 and restimulated cells, mRNA levels of Brca1 ( C ) and Brca2 ( D ) were analyzed. Maximal fold-changes were calculated as the ratio of the highest to lowest expression levels. Averages from two technical replicates each from four independent cell lines for each cell variant are shown. Expression was normalized to WT proliferating ( A , B ) or to maximum expression of WT ( C , D ).

Article Snippet: RPE-1, NIH3T3, HFF, and T98G cells (DSMZ, Braunschweig, Germany) as well as HCT116 wild-type, p53 -/- and p21 -/- cells [ ] were grown in DMEM (Lonza) supplemented with 10% FCS and penicillin/streptomycin and maintained at 37 °C and 10% CO 2 .

Techniques: Expressing, Variant Assay

Journal: Cell Death and Differentiation

Article Title: BRCA1 and BRCA2 gene expression: p53- and cell cycle-dependent repression requires RB and DREAM

doi: 10.1038/s41418-025-01566-9

Figure Lengend Snippet: NIH3T3 cells were transfected with luciferase reporter constructs of the wild-type human promoters (wt) and mutant promoters for potential transcription factor binding sites (E2F-A, E2F-B, and A/B) of ( A ) BRCA1 , with A/B representing a mutant of E2F-A and E2F-B or ( B ) wt and mutant promoter construct of BRCA2 together with a Renilla luciferase control reporter plasmid. Cells were synchronized in G 0 by serum starvation, stimulated to re-enter the cell cycle be serum addition and collected after 24 h. Relative luciferase activity is given (Mean ± SEM, n = 3–4; two-tailed unpaired t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: RPE-1, NIH3T3, HFF, and T98G cells (DSMZ, Braunschweig, Germany) as well as HCT116 wild-type, p53 -/- and p21 -/- cells [ ] were grown in DMEM (Lonza) supplemented with 10% FCS and penicillin/streptomycin and maintained at 37 °C and 10% CO 2 .

Techniques: Transfection, Luciferase, Construct, Mutagenesis, Binding Assay, Control, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: BRCA1 and BRCA2 gene expression: p53- and cell cycle-dependent repression requires RB and DREAM

doi: 10.1038/s41418-025-01566-9

Figure Lengend Snippet: A Chromatin immunoprecipitations (ChIPs) were performed with cross-linked chromatin from serum-starved (0 h) or restimulated (22 h) T98G cells. Antibodies targeted E2F4, E2F1, E2F3, or B-MYB. A non-targeting antibody (IgG) and the promoter of the GAPDHS gene served as a negative control. The BRCA1 and BRCA2 promoters were detected by real-time qPCR. All signals are given relative to the input DNA signal. B ChIPs were performed with cross-linked chromatin from starved (0 h) or restimulated (22 h) RPE-1 cells. Antibodies targeted E2F4, LIN9, LIN37, A-MYB, or B-MYB. A non-targeting antibody (IgG) and the promoter of the GAPDHS gene served as a negative control. The BRCA1 and BRCA2 promoters were detected by real-time qPCR. All signals are given relative to the input DNA signal. A , B Mean ± SD; two-way ANOVA; n = 3; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. C Nuclear extracts of density-arrested RPE-1 cells and cells restimulated for 20 h were employed for DNA affinity purification using biotinylated wt and mutant (E2F-A, E2F-B, and A/B) BRCA1 promoter probes and a promoter probe of the late cell cycle gene Cyclin B2 as well as a mouse promoter probe of the early cell cycle gene Dhfr . As a negative control, a fragment of the mouse Gapdhs promoter (neg. Ctrl.) was used. D DREAM components (p130, E2f4, Lin37, Lin54, and Lin9) were purified from nuclear extracts of density-arrested NIH3T3 mouse cells by DNA affinity purification and detected by Western blot. Binding to the human BRCA2 wild-type promoter probe (wt) was compared with binding to mutant probes (E2F-A, E2F-B, and A/B). Background protein binding was determined with a probe of the mouse cyclin B2 CHR mutant promoter (neg. Ctrl.). E p130, RB, E2F4, and LIN37 were purified from nuclear extracts of proliferating HCT116 human cells by DNA affinity purification and detected by Western blot. Binding to the wild-type promoter probe (wt) was compared with binding to E2F site mutant probes (E2F-A, E2F-B, and A/B) as well as binding to the promoter of the late cell cycle gene Cyclin B2 and the mouse promoter of the early cell cycle gene Dhfr . As a negative control, binding to a fragment of the mouse Gapdhs promoter was tested (neg. Ctrl.).

Article Snippet: RPE-1, NIH3T3, HFF, and T98G cells (DSMZ, Braunschweig, Germany) as well as HCT116 wild-type, p53 -/- and p21 -/- cells [ ] were grown in DMEM (Lonza) supplemented with 10% FCS and penicillin/streptomycin and maintained at 37 °C and 10% CO 2 .

Techniques: Negative Control, Affinity Purification, Mutagenesis, Purification, Western Blot, Binding Assay, Protein Binding

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

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. 2. p35C/EBPb and p20C/EBPb contribute to SRE BP DNA binding activity. (A) Nuclear extract from NIH 3T3 cells was fractionated on an SDS– 12% polyacrylamide gel and transferred to an Immobilon-P membrane. One lane was processed for Western blotting with anti-C/EBPb peptide antibody, while the separated proteins with apparent molecular masses between 66 and 14 kDa were renatured in elution solution from a parallel lane 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 500 ng of poly(dI-dC): poly(dI-dC) as described in Materials and Methods. A scale of apparent molec- ular masses in kilodaltons, based on protein standards run in parallel on the SDS gel and detected on the Immobilon-P filter by Ponceau S staining, is shown above the lanes. (B) Ten microliters of the SDS-sized fractions from the lanes indicated in panel A was preincubated for 10 min at 378C with 1 ml of the indicated peptide antibodies (Santa Cruz Biotechnology) or 1 ml of 1-mg/ml BSA in the presence of 20 ng of aprotinin. Protein samples were then 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. This figure was prepared with a Mirror 800 color scan- ner 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: Binding Assay, Activity Assay, Membrane, Western Blot, Gel Shift, Labeling, SDS-Gel, Staining

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. 3. Effect of C/EBPb antibodies on SRE BP DNA binding activity in nuclear extract. 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. Specific SRF-DNA or C/EBPb protein-DNA complexes are indicated. The radioactivity levels in specific protein-DNA complexes on the dried gel were quantified with a Bio-Rad molecular imager. The graph represents the decrease in SRE BP complex formation with antibody or preimmune serum incubation. The amount of SRE BP complex formed when nuclear extract was incubated with BSA was assigned a value of 100%. Data are the averages of two separate experiments for the E12 antibody, three separate experiments for the guinea pig antibody and preimmune serum, and five separate experiments for the Santa Cruz Biotechnology C/EBP antibodies and BSA. The average error in the determinations was 14%. 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: Binding Assay, Activity Assay, Labeling, Radioactivity, Incubation

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. 4. Regulation of the cfos SRE by p20C/EBPb and p35C/EBPb. NIH 3T3 cells were transfected 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-LAP (p35) (1LAP), 0.5 mg of CMV-LIP(p20) (1LIP), or 1 mg of CMV-LAP and 0.5 mg of CMV-LIP (1LAP/LIP). Total DNA in each transfection was adjusted to 15 mg with pUC19 DNA. Twenty-four hours after transfection, cells were serum deprived 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 harvested 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.

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: Transfection, Plasmid Preparation, Activity Assay

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. 5. Regulation of the cfos SRE by C/EBPb is independent of TCF binding. NIH 3T3 cells were transfected as described in Materials and Methods with 10 mg of a TCF mutant SRE CAT reporter gene (solid symbols) and either 2.5 mg of CMV-4 vector (circles), 2.5 mg of CMV-LAP (p35) (squares, solid lines), 0.5 mg of CMV-LIP(p20) (triangles), or 2.5 mg of CMV–NF-IL6 (squares, broken line). Parallel experiments with the wild-type SRE CAT reporter gene are denoted by open symbols. CAT activity is expressed as fold increase, calcu- lated as described in the legend to Fig. 4. Error bars have been omitted for clarity; however, the average error was 61.6-fold for the CMV-4 and CMV-LIP transfections, 611-fold for the CMV-LAP transfections, and 617-fold for the CMV–NF-IL6 transfections.

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: Binding Assay, Transfection, Mutagenesis, Plasmid Preparation, Activity Assay

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. 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.

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: Plasmid Preparation, Transfection, Activity Assay

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. 9. SRE BP binding to SRE mutant DNAs. Gel shift assays were per- formed as described in Materials and Methods using buffer I conditions with approximately 8 mg of nuclear extract from NIH 3T3 cells and 1 ng of 32P-labeled SRE DNA in the presence of 2 mg of poly(dI-dC):poly(dI-dC) and the indicated amount of competitor SRE mutant oligonucleotide (solid bars) or wild-type SRE DNA (open bars). Radioactivity levels in the SRE BP-DNA complexes on the dried gel were quantified with a Bio-Rad molecular imager. The ratio of DNA bound in the presence versus the absence of competitor DNA for each concen- tration of competitor DNA added to the binding reaction mixtures was calcu- lated and plotted as shown. Values are the averages of duplicate determinations. Sequence changes between the wild type and FSS (A), SRE.M (B), and SRE.LP (C) mutants are shown at the left. Complete sequences of the wild-type and mutant SRE oligonucleotides used in the competitions are given in Materials and Methods. (D) A representative electrophoretic mobility shift assay used to obtain the graphical data presented in panel B is shown. Only the SRF-DNA and SRE BP-DNA complexes are shown. The amount of SRE BP complex plotted in panels A to C represents the summation of the radioactivity levels in each of the three discernible bands within the SRE BP complex, although similar results were obtained by considering each of the three SRE BP bands individually. 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: Binding Assay, Mutagenesis, Gel Shift, Labeling, Radioactivity, Sequencing, Electrophoretic Mobility Shift Assay

Journal: bioRxiv

Article Title: Lipid moieties of sonic hedgehog are important for interaction with its inhibitor, WIF1

doi: 10.64898/2026.02.23.707386

Figure Lengend Snippet: Comparison of the signaling activities of rhShh_8908-SH, rhShh_1314-SH and rhShh_1845-SH in the Gli Reporter-NIH3T3 cell line reporter assay. Panel A: rhShh_8908-SH expressed in HEK293 cells was applied at 0–600 nM. EC 50 = 34.22 ± 2.25 nM. Panel B: rhShh_1314-SH expressed in E. coli was applied at 0–2400 nM. EC 50 = 64.81 ± 1.29 nM. Panel C: rhShh_1845-SH expressed in E. coli was applied at 0–2400 nM. EC 50 = 86.09 ± 1.11 nM. In all assays, Gli Reporter-NIH3T3 cells were grown to confluency, the culture medium was removed, and cells were treated with 50 μL aliquots of the indicated Shh concentrations. Data represent one experiment performed in triplicate and are expressed as fold induction relative to luminescence at 0 nM Shh.

Article Snippet: The ONE-StepTM Luciferase Assay System and the Gli Reporter-NIH3T3 cell line were purchased from BPS Bioscience (Inc., San Diego, CA, USA).

Techniques: Comparison, Reporter Assay

Journal: bioRxiv

Article Title: Lipid moieties of sonic hedgehog are important for interaction with its inhibitor, WIF1

doi: 10.64898/2026.02.23.707386

Figure Lengend Snippet: Inhibition of the signaling activities of rhShh_8908-SH, rhShh_1314-SH and rhShh_1845-SH proteins by human WIF1 protein in the Gli Reporter-NIH3T3 cell line reporter assay. Panel A: Inhibition of human rhShh_8908-SH expressed in HEK293 cells (15 nM) by rhWIF1 (0–1000 nM); EC 50 = 3.78 ± 0.13 nM. Panel B: Inhibition of rhShh_1314-SH expressed in E. coli (15 nM) by rhWIF1 (0–1200 nM); EC 50 = 6.83 ± 0.89 nM. Panel C: Inhibition of rhShh_1845-SH expressed in E. coli (15 nM) by rhWIF1 (0–1200 nM); EC 50 = 15.71 ± 1.75 nM. In all assays, Gli Reporter-NIH3T3 cells were grown to confluency, culture medium was removed, and cells were treated with 50 μL aliquots of rhShh preincubated for 5 min with rhWIF1. Data represent representative experiments performed in triplicate (B, C) or quadruplicate (A) and are expressed as fraction of luminescence relative to 0 nM rhWIF1.

Article Snippet: The ONE-StepTM Luciferase Assay System and the Gli Reporter-NIH3T3 cell line were purchased from BPS Bioscience (Inc., San Diego, CA, USA).

Techniques: Inhibition, Reporter Assay