Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: p107 and p130 are inhibitors of cdk2 activity in vivo. (A) p107 expression is modestly induced in a tetracycline-repressible cell line by removal of tetracycline. +, p107 induction; −, expression was not induced. (B) Induction of p107 levels shown in panel A causes significant reduction in histone H1 kinase activity. Results of a representative experiment are shown. (C) Quantitation of kinase activity shown in panel B.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Activity Assay, In Vivo, Expressing, Quantitation Assay

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Inhibition of cyclin A (cyc A)- and cyclin E-cdk2 by p107 and p21 in vitro. (A) Recombinant proteins used in the in vitro assays. p107, pRB, N385, cyclin A-cdk2, and cyclin E-cdk2 were purified from insect cells infected with recombinant baculoviruses. GST-tagged Δ10N was purified from bacteria as described in Materials and Methods. Equal amounts of the indicated proteins were electrophoresed and visualized by silver staining. The sizes of molecular mass markers (lane M) (in kilodaltons) are indicated on the left. (B to D) Histone H1 kinase assays were performed with purified p21 from Escherichia coli or purified p107 (see Materials and Methods). Increasing concentrations of p21 (0.05 to 74 nM), p107 (0.01 to 30 nM), or prephosphorylated p107 (0.01 to 30 nM) were used as indicated. The relative levels of phosphorylated histone H1 were determined with a PhosphorImager. Histone H1 phosphorylation by cyclin A-cdk2 in the absence of an inhibitor was given a value of 100. Calculated average Ki values are indicated. (E) Histone H1 kinase assays comparing p21 and p107 as inhibitors of cyclin E-cdk2. Increasing concentrations of p107 (0.7 to 42 nM) (lanes 2 to 10) or p21 (0.7 to 42 nM) (lanes 12 to 20) were incubated with 1 ng of cyclin E-cdk2. For each set of reactions, a kinase-alone control (lanes −) was included. (F) The relative levels of phosphorylated histone H1 were determined as for panels B to D. Each value represents the mean and standard error of the mean for four independent experiments.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Inhibition, In Vitro, Recombinant, Purification, Infection, Silver Staining, Incubation

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: p107 and pRB as inhibitor and substrate, respectively, of cyclin A (cyc A)-cdk2. (A) p107 was allowed to bind cyclin A-cdk2 in the absence or presence of 1 μM ATP for 30 min at 37°C. Glutathione-agarose precipitation of GST-cyclin A-cdk2 was carried out as described in Materials and Methods, followed by immunoblot analysis of p107. The input amount of p107 is shown in lane 1. (B) p107 (lanes 3, 4, 9, and 10) and pRB (lanes 5, 6, 11, and 12) were incubated in a kinase reaction without histone H1 and with [γ-32P]ATP. After 30 min at 37°C, half of the reaction mixture (lanes 1 to 6) was taken and boiled in SDS sample buffer, while the other half (lanes 7 to 12) was incubated with glutathione-agarose beads and precipitated as described in Materials and Methods. (C) Cyclin A-cdk2 kinase reactions in the presence of 20 ng of p107 (lanes 2 and 3), 60 ng of N385 (lanes 4 and 5), or 20 ng of pRB (lanes 6 and 7). (D) An extended titration comparing the effects of equal amounts of pRB and p107 on cyclin A-cdk2 kinase activity. The amount of either protein is indicated below the autoradiogram. (E) Quantitation of the data shown in panel D. The relative percentage of kinase activity was determined in each case by the amount of 32P incorporated into histone H1, as measured with a PhosphorImager.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Western Blot, Incubation, Titration, Activity Assay, Quantitation Assay

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Inhibition of cyclin A (cyc A)-cdk2 substrates. (A) Kinase assays were performed in the absence (left) or presence (right) of 20 ng of p107 (previously phosphorylated). One hundred nanograms of the indicated purified proteins was added to the reaction mixture, with the exception of histone H1, for which 1.25 μg was used. (B) The amount of phosphorylation of each protein was quantified with a PhosphorImager, and the level of phosphorylation of each protein in the absence of p107 was set to 100%. The values represent the means and standard errors of the means for four different experiments.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Inhibition, Purification

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Peptide binding competition of p107 to cyclin A (Cyc A)-cdk2. (A to C) Western blot analysis carried out after GST precipitation of GST-cyclin A-cdk2 shows the capacity of peptides p107S and p107N, but not p107N-mut, to compete the binding of p107 to cyclin A-cdk2. The concentrations of peptides range from 2 nM to 5 μM. The amount of cyclin A retained was not affected by addition of peptide and was included as a control. (D) Western blots were densitometrically scanned and quantified by utilizing NIH Image 1.61 software. The values indicate the means and standard errors of the means for three independent experiments. (E) Cyclin A-cdk2 kinase reaction. The peptides p107S and p107N, but not p107N-mut, were able to partially reverse the inhibition of cyclin A-cdk2 by p107 or Δ10N. The peptides had no effect on the kinase reaction by themselves (compare lane 1 to lanes 2 to 4).

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Binding Assay, Western Blot, Software, Inhibition

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Inhibition of cyclin A (cyc A)- and cyclin E-cdk2 by the amino terminus of p107. (A) Schematic diagram of p107, N385, and Δ10N proteins. Shaded rectangles with A and B denote subdomains of the carboxy-terminal region conserved with pRB and p130, and black rectangles indicate potential amino-terminal and spacer region cyclin-binding domains. (B) Kinase reactions comparing equal amounts (approximately 20 ng) of p107 (lanes 2 and 6), Δ10N (lanes 3 and 7), and N385 (lanes 4 and 8). All of the reaction mixtures contained 1.25 μg of histone H1 as a substrate for cyclin A-cdk2 (lanes 1 to 4) or cyclin E-cdk2 (lanes 5 to 8), and each had been preincubated at room temperature in the presence of unlabeled 1 μM ATP. The positions of phosphorylated histone H1 and p107 proteins are indicated at the left.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Inhibition, Binding Assay

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: An amino-terminal region in p107 is important for growth suppression. (A) Schematic alignment of cyclin-binding domains found in the p21-p27-p57 and p107-p130 families of proteins. Previously identified sequences found in the spacer regions (S) of p107 and p130 are indicated. Homologous amino-terminal sequences (N) found in p107, p130, and the Drosophila pRB-related factor, RBF, are shown. (B) Diagram of p107 constructs used in transfection assays. Symbols used are as indicated in the legend to Fig. ​Fig.5.5. Deletions and triple alanine point mutations (asterisks) are indicated. (C) Growth arrest of C33A cells mediated by p107 as determined by FACS analysis (see Materials and Methods). The ordinate shows the absolute difference in percentage of cells in the G1 phase of the cell cycle. Values represent averages for at least three independent experiments. (D) Growth arrest of Saos-2 cells by p107 L19 derivatives.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Binding Assay, Construct, Transfection

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Mutations in p107 abolish binding to cyclins A and E. Each of the indicated proteins was expressed in C33A cells by transfection, and whole-cell extracts were immunoprecipitated with antibodies against p107 or the HA tag (which recognize the carboxy-terminal tag on p107 constructs). Immunoprecipitates were electrophoresed and subjected to immunoblotting with the indicated antibodies. A negative control transfection with empty expression vector (lane −) is indicated.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Construct, Western Blot, Negative Control, Expressing, Plasmid Preparation

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Mutations in p130 similar to those introduced into p107 also abolish cyclin binding and diminish growth-suppressive activity. (A) Schematic of p130 mutations. Symbols are described in legend to Fig. ​Fig.6.6. (B) Cyclin-binding activity of transfected p130 constructs. Endogenous cyclins were coimmunoprecipitated with p130 by using anti-p130 antibodies as described in the legend to Fig. ​Fig.7.7. (C) Growth arrest of C33A cells as determined in Fig. ​Fig.6C.6C. The transfected p130 plasmids are indicated below the graph, and results represent averages for several independent experiments.

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Binding Assay, Activity Assay, Transfection, Construct

Journal:

Article Title: Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

doi:

Figure Lengend Snippet: Mutation of the p107 amino-terminal cyclin-binding domain abolishes kinase inhibition and binding. Cyclin A (cyc A) (A) and cyclin E (B) bind Δ10N and N385, albeit less efficiently than full-length p107, but do not bind the mutant Δ10N-AAA. (A) Western blot analysis of cyclin A bound to p107 derivatives. Thrombin-cleaved cyclin A, devoid of a GST tag, was tested for binding to GST alone (lane 2), Δ10N (lane 3), Δ10N-AAA (lane 4), p107 (lane 5), and N385 (lane 6). The input amount of cyclin A is shown in lane 1. (B) Western blot analysis of cyclin E bound to p107 derivatives. Thrombin-cleaved cyclin E, devoid of a GST tag, was tested for binding to GST alone (lane 2), Δ10NAAA (lane 3), N385 (lane 4), p107 (lane 5), and Δ10N (lane 6). The cyclin E input is indicated in lane 1. In each case, precipitation of N385 and p107 was performed with a human papillomavirus E7 peptide linked to Sepharose beads, and precipitation of Δ10N and Δ10N-AAA was performed with glutathione-agarose beads as described in Materials and Methods. (C) Kinase reactions comparing equal amounts of Δ10N-AAA (lanes 3 and 4), Δ10N (lanes 5 and 6), or p107 (lanes 7 and 8).

Article Snippet: Polyclonal antibodies against p107 (C-18), cyclin A (H-432), cdk2 (M2), and p130 (C-20) were obtained from Santa Cruz Biotechnology, and anti-influenza virus hemagglutinin (HA) antibody 12CA5 was obtained from Berkeley Antibody Co.

Techniques: Mutagenesis, Binding Assay, Inhibition, Western Blot

Journal:

Article Title: Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

doi: 10.1128/MCB.23.3.1095-1111.2003

Figure Lengend Snippet: Proliferative effects of HRG require PR and p42/p44 MAPK activation. C4HD (A) and T47D (B) cells were incubated for 48 h in medium with ChFCS supplemented, as indicated, with RU486, 10 nM MPA, MPA-RU486, HRG, HRG-RU486, HRG-ASODN or -SODN plus ErbB-2, or HRG-PD98059. C4HD cells were also treated with HRG plus increasing concentrations (1, 5, and 10 μg/ml) of either ErbB-3 monoclonal antibody (Ab-5) or ErbB-4 monoclonal antibody (Ab-3) and HRG plus preimmune mouse serum (NMS). Incorporation of [3H]thymidine was used as a measure of DNA synthesis. Data are presented as means ± the standard deviation (SD). Significance for columns b versus a, columns c versus b, columns d versus a, and columns e versus d, P < 0.001. The experiments shown are representative of a total of five for each cell type.

Article Snippet: Membranes were blocked with PBS-0.1% Tween 20 and immunoblotted with the following antibodies: ErbB-2 rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotechnology, Santa Cruz, Calif.), ErbB-3 rabbit polyclonal antibody C-17 (Santa Cruz), ErbB-4 rabbit polyclonal antibody C-18 (Santa Cruz), ErbB-4/HER-4 oncoprotein Ab-2 rabbit polyclonal antibody (Neomarkers), anti-total p42/p44 MAPK (C-14; Santa Cruz) , anti-phospho p42/p44 MAPK (E-4 Santa Cruz), and antihemagglutinin (anti-HA) antibody HA.11 (Berkeley Antibody Co., Berkeley, Calif.).

Techniques: Activation Assay, Incubation, DNA Synthesis, Standard Deviation

Journal:

Article Title: Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

doi: 10.1128/MCB.23.3.1095-1111.2003

Figure Lengend Snippet: Blockage of ErbB-2 expression, ErbB-3 activation, or MAPK activity abolishes HRG capacity to induce PR binding to a PRE. (A) C4HD and T47D cells were treated for 2 h at 37°C with 10 nM MPA or HRG at 20 ng/ml or remained untreated and growing in ChFCS. ErbB-2 expression was blocked by pretreatment of both cell types for 48 h with 2 μM ASODN to ErbB-2 before the 2-h treatment with HRG. As a control, cells were preincubated with 2 μM SODN. MEK1/MAPK activity was abolished by preincubating the cells for 30 min with 10 μM PD98059 before treatment with HRG for 2 h. C4HD cells were also pretreated with 10 μg of ErbB-3 monoclonal antibody (Ab-5), ErbB-4 monoclonal antibody (Ab-3), or preimmune mouse serum (NMS)/ml before treatment with HRG for 2 h. Aliquots of nuclear extracts (4 fmol of PR) were incubated with a [32P]PRE oligonucleotide and subjected to an EMSA. (B) C4HD and T47D cells were incubated for 2 h with HRG at 20 ng/ml or preincubated for 48 h with 2 μM ErbB-2 ASODN or SODN and then treated for 2 h with HRG as described above. A total of 80 μg of protein from cell lysates was electrophoresed and immunoblotted for ErbB-2. Densitometric analysis of an ErbB-2 band expressed as percentages of the control value, i.e., cells growing in 20 ng of HRG/ml, yielded 32% for C4HD cells and 26% for T47D cells treated with 2 μM ASODN. No significant differences were found in the densitometric values of ErbB-2 bands between control cells and cells treated with 2 μM SODN. Autoradiograms from representative experiments of a total of three performed for each cell type are shown. (C to F) To evaluate the role of blocking antibodies on HRG-induced ErbB-3 and ErbB-4 tyrosine phosphoryation, C4HD cells were preincubated with 10 μg of either a ErbB-3 mouse monoclonal antibody (Ab-5) or a ErbB-4 mouse monoclonal antibody (Ab-3)/ml prior to HRG treatment for 10 min. As a control, cells were also incubated with preimmune normal mouse serum (NMS). ErbB-3 and ErbB-4 were immunoprecipitated as described in Materials and Methods, and immunocomplexes were subjected to SDS-PAGE (7.5% gel) and analyzed by Western blotting with an anti-P-Tyr monoclonal antibody (C and E). Identical aliquots of each immunoprecipitate were subjected to immunoblot analysis with anti-ErbB-3 (D) or ErbB-4 (F) antibodies to verify that nearly equal amounts of immunoprecipitated proteins were loaded. W, Western blot; IP, immunoprecipitation. (G) C4HD and T47D cells were incubated for 10 min with HRG at 20 ng/ml or were left untreated and growing in ChFCS. Blockage of MAPK activity was accomplished by preincubating the cells for 30 min with 10 μM PD98059 before stimulation with HRG. Cell lysates were immunoprecipitated by using an anti-p42/p44 MAPK antibody. The immunoprecipitates were assayed for MAPK activity with MBP as the substrate. (H) As loading control, identical aliquots of each immunoprecipitate assayed in panel G were subjected to immunoblot analysis with the anti-p42/p44 MAPK antibody. (I) C4HD and T47D cells were treated as described in panel G and, in addition, C4HD were treated with MPA for 10 min or were preincubated for 30 min with 10 μM PD98059 before stimulation with MPA. A total of 100 μg of protein from cell lysates was electrophoresed on 12% SDS-gels and immunoblotted with an anti-phospho p42/p44 MAPK antibody. (J) The membrane shown in panel I was stripped and hybridized with an antibody anti-total p42/p44 MAPK antibody.

Article Snippet: Membranes were blocked with PBS-0.1% Tween 20 and immunoblotted with the following antibodies: ErbB-2 rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotechnology, Santa Cruz, Calif.), ErbB-3 rabbit polyclonal antibody C-17 (Santa Cruz), ErbB-4 rabbit polyclonal antibody C-18 (Santa Cruz), ErbB-4/HER-4 oncoprotein Ab-2 rabbit polyclonal antibody (Neomarkers), anti-total p42/p44 MAPK (C-14; Santa Cruz) , anti-phospho p42/p44 MAPK (E-4 Santa Cruz), and antihemagglutinin (anti-HA) antibody HA.11 (Berkeley Antibody Co., Berkeley, Calif.).

Techniques: Expressing, Activation Assay, Activity Assay, Binding Assay, Incubation, Blocking Assay, Immunoprecipitation, SDS Page, Western Blot

Journal:

Article Title: Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

doi: 10.1128/MCB.23.3.1095-1111.2003

Figure Lengend Snippet: HRG induces transcriptional activity of PR. C4HD (A) and T47D (B) cells were transiently transfected with 4 μg of a PRE2-tk-CAT reporter plasmid/well and with 4 μg of a CMV-β-Gal expression vector/well as an internal control. C4HD cells were also transfected with a tk-CAT reporter lacking a PRE. Cells were then treated with MPA, MPA-RU486, HRG, and HRG-RU486 at 37°C for 48 h for C4HD cells or for 24 h for T47D cells or were left untreated and growing in ChFCS. To study the effect of ErbB-2 expression blockage, 2 μM ErbB-2 ASODN or 2 μM SODN was added, along with HRG, during the 48-h treatment. To inhibit MAPK activation, cells were preincubated with 10 μM PD98059 before HRG treatment. C4HD cells were also pretreated with 10 μg of ErbB-3 monoclonal antibody (Ab-5) or ErbB-4 monoclonal antibody (Ab-3)/ml or preimmune mouse serum before treatment with HRG. Cells were harvested and lysed, and the CAT and β-galactosidase activities were measured as described in Materials and Methods. The results are presented as the percent activation, where 100% represents the maximal activity of PR in the presence of MPA. The data shown represent the means of six independent experiments ± the SEM. Significance for columns b versus a, columns c versus b, columns d versus a, and columns e versus d, P < 0.001.

Article Snippet: Membranes were blocked with PBS-0.1% Tween 20 and immunoblotted with the following antibodies: ErbB-2 rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotechnology, Santa Cruz, Calif.), ErbB-3 rabbit polyclonal antibody C-17 (Santa Cruz), ErbB-4 rabbit polyclonal antibody C-18 (Santa Cruz), ErbB-4/HER-4 oncoprotein Ab-2 rabbit polyclonal antibody (Neomarkers), anti-total p42/p44 MAPK (C-14; Santa Cruz) , anti-phospho p42/p44 MAPK (E-4 Santa Cruz), and antihemagglutinin (anti-HA) antibody HA.11 (Berkeley Antibody Co., Berkeley, Calif.).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Activation Assay

Journal: Neuron

Article Title: The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

doi: 10.1016/s0896-6273(01)00553-0

Figure Lengend Snippet: Figure 5. Localization of EphB2 and Ephrin Expression In Vivo and In Vitro

Article Snippet: Histochemistry/Immunohistochemistry (IHC) Sections were blocked in 5% normal goat serum, 0.2% Tween-20Activity-Dependent Regulation of EphB2 in PBS for 1 hr at RT followed by incubation in primary antibodyat Neural Synapses 4 C. Primary antisera were as follows: Calbindin D (Sigma) 1:400,The results obtained for hippocampal neurons both in calretinin (Chemicon) 1:400, parvalbumin (Sigma) 1:600, mGluR5vitro and in vivo demonstrate that in addition to regulat(Upstate Biotechnology) 1:1000, Ephrin B1 (C-18 Santa Cruz) 1:1000, ing NMDA-dependent synaptic responses, EphB2 can and GluR1a (Upstate Biotechnology) 1:3000.

Techniques: Expressing, In Vivo, In Vitro

Journal: The Journal of investigative dermatology

Article Title: Expression patterns of the transcription factor AP-2alpha during hair follicle morphogenesis and cycling.

doi: 10.1046/j.1523-1747.2003.12319.x

Figure Lengend Snippet: Figure1. Expression of AP-2a (red £uorescence) during di¡erent stages of HF morphogenesis in neonatal mouse skin (days 0^5 postpartum). Cryostat sections (10 mm) were incubated with primary rabbit antihuman AP-2a polyclonal antibodies and then treated with rhodamine-conjugated F(ab)2 fragments of a goat antirabbit IgG. (A) Stage 1 of HF morphogenesis. High AP-2a IR is seen in the epithelial placode. Mesenchymal condensation beneath the follicle plug is slightly positive (arrow). (B)^(C) Stage 2. Some suprabasal epidermal keratinocytes above the placode are highly positive as well (arrow- heads). (D) Stage 3. The entire hair plug becomes AP-2a positive while engul¢ng the FP (arrow). (E) Stage 4.With progression of the downward growth of the HF, AP-2a expression declined in the lower portion but remained high in the follicle infundibulum. (F) Stage 5. Three zones of AP-2a IR are formed in the HF: the uppermost HF portion (infundibulum) (1), the zone of sebocyte di¡erentiation (2), and a newly formed morphogenic zone that gives rise to the IRS (3). (G) With initiation of formation of ascending layers of HF, AP-2a expression is most prominent in the IRS cone (white arrowhead). (H) The early formation of the hair shaft and invasion of the HF into the dermis (stages 5^6 of morphogenesis) were associated with an increase of AP-2a IR in FP cells. (I) The sebaceous glands, the epithelial lining of the HF infundibulum, and the basal layer of interfollicular epidermis remained persistently AP-2a positive. be, basal layer of epidermis; ORS, outer root sheath; sg, sebaceous gland. Scale bars: (A)^(E), (H) 23 mm; (F), (G), (I) 35 mm.

Article Snippet: After the overnight incubation (room temperature) with primary rabbit antihuman AP-2a polyclonal antibodies (sc-184, clone C-18; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500, the sections were treated with rhodamine-conjugated F(ab)2 fragments of a goat antirabbit IgG (Jackson ImmunoResearch,West Grove, PA) at a dilution of 1:200 (1 h; 371C), counterstained with Hoechst 33342 dye (10 mg per ml) (Sigma, St. Louis, MO), and mounted with Vectashield H-1000 (Vector, Burlingame, CA).

Techniques: Expressing, Incubation

Journal:

Article Title: RBP1 Recruits Both Histone Deacetylase-Dependent and -Independent Repression Activities to Retinoblastoma Family Proteins

doi:

Figure Lengend Snippet: Endogenous interactions of pRB (A) and p107 (B) with HDACs. (A) Immunoprecipitations were done in lysates from either H1299 or 293T cells. Plates (100 mm) of both cell types were lysed in low-stringency buffer. Cell extracts were incubated with 1 μg of the indicated antisera and 30 μl of a 50% slurry of protein G-Sepharose for at least 12 h. Immunoprecipitated complexes were washed six times with the 150 mM low-stringency buffer and eluted by boiling with 2× sample buffer. Eluted proteins were subjected to SDS-PAGE with a 6% polyacrylamide gel. The presence of pRB was detected with monoclonal antibody (G3-245) against pRB (PharMingen). (B) Binding studies similar to those described for panel A were done, but coimmunoprecipitation of p107 was detected with a rabbit polyclonal antibody (C-18) against p107 (α-107) (Santa Cruz).

Article Snippet: The presence of pRB was detected with monoclonal antibody (G3-245) against pRB (PharMingen). (B) Binding studies similar to those described for panel A were done, but coimmunoprecipitation of p107 was detected with a rabbit polyclonal antibody (C-18) against p107 (α-107) (Santa Cruz).

Techniques: Incubation, Immunoprecipitation, SDS Page, Binding Assay