Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 4. Effects of estradiol on cyclin protein expression. The experimental design was described in Fig. 2. Representative Western blots are shown for G1 phase cyclins (D1, D3, E) (A) and S/G2M phase cyclins (A, B1) (B). Controls for cyclin E are as described in Fig. 2.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 5. Estrogen induction of cyclin D1 and cyclin D3 mRNA expression. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, total cellular RNA was harvested. A, representative North- ern blots are shown for cyclin D1 and D3 mRNA from estradiol-treated and control cells. Arrows indicate the 4.5- and 1.5-kb cyclin D1 transcripts. B, graphical pres- entation of temporal changes in mRNA (open symbols) and protein (solid symbols, mean of two experiments) for cyclin D1 (left) and cyclin D3 (right).

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 8. Activation of Cdk4 and Cdk2 following estrogen treatment. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immunoprecipitated with antibodies to either Cdk4, cyclin E, or Cdk2, and then the kinase activity of the immunoprecipitates was determined by phosphorylation of GST-pRB773–923

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Immunoprecipitation, Activity Assay, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 9. Composition of cyclin D1-as- sociated and cyclin E-associated complexes. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immuno- precipitated with anti-cyclin D1 anti- serum or anti-cyclin E antibodies, and then these immunoprecipitates were sep- arated by SDS-PAGE and transferred to nitrocellulose membranes. A, cyclin D1 antiserum immunoprecipitates. The same filter was sequentially Western blotted for cyclin D1, Cdk4, p21, and p27. A rep- resentative blot is shown for each. B, rel- ative levels of cyclin D1 (E), Cdk4 (G), p21 (M), and p27 (f) were determined by den- sitometry and are expressed relative to the vehicle treated controls. Points repre- sent the mean of two separate experi- ments. C, cyclin E immunoprecipitates. The same filter was sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Control, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 10. Estrogen decreases inhibi- tory activity toward cyclin E-Cdk2. A, lysates were prepared from cells that were pretreated with antiestrogen and then treated with E2 (1) or vehicle (2). Active cyclin E-Cdk2 complexes that had been prepared from baculovirus-infected Sf9 cells were incubated with either lysis buffer only (labeled input) or with cell lysates. Lysates were also immunode- pleted with either anti-p27 antibodies, anti-p21 antibodies, or both and then in- cubated with recombinant cyclin E-Cdk2 complexes. Recombinant cyclin E-Cdk2 complexes were then recovered and as- sayed for histone (H1) kinase activity. The percentage of the input activity, de- fined as 100%, is also shown numerically below the autoradiograph. The same sam- ples were electrophoresed on a duplicate SDS-PAGE gel and then Western blotted for p21 and p27 to assess the binding of these proteins to recombinant cyclin E- Cdk2. B, the experiment described for panel A was repeated using either boiled lysis buffer (labeled input) or boiled ly- sates from cells treated with estradiol (1) or vehicle (2). C, MCF-7 cells were pre- treated with 10 nM ICI 182780 for 48 h and then treated with 100 nM estradiol or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were pre- pared and assayed for cyclin E-Cdk2 in- hibitory activity as described for panel A.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activity Assay, Infection, Incubation, Lysis, Labeling, Recombinant, Autoradiography, SDS Page, Western Blot, Binding Assay, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 11. Cyclin E-Cdk2 activation is accompanied by loss of CDK inhibitor association and Cdk2 Thr-160 phosphorylation. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. Whole cell lysates were prepared either 8 h after estrogen (8 h E2) or from control cells (ICI 182780). Lysates were then fractionated on a Superose 12 gel filtration column. A, fractions were precipitated with acetone and Western blot- ted for cyclin E (top panels) or assayed for cyclin E-Cdk2 histone (H1) kinase activity (bottom panel). The relative levels of cyclin E protein (E, G) and cyclin E-Cdk2 activity (M, f) in lysates from antiestrogen- pretreated cells and cells treated with estradiol for 8 h were determined by densitometry and are represented graphically. The elution of mark- ers of known molecular weight (ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa) are indicated at the top of the graph. B, fractions 19 and 24 from the 8 h E2 lysate were immunoprecipitated with an anti- cyclin E antibody, and the immunoprecipitates were electrophoresed on a SDS-PAGE gel and transferred to a nitrocellulose filter. The same filter was then sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Control, Filtration, Western Blot, Activity Assay, Molecular Weight, Immunoprecipitation, SDS Page

Journal: The Journal of biological chemistry

Article Title: Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.

doi: 10.1074/jbc.274.19.13198

Figure Lengend Snippet: FIG. 2. Binding studies of GST-PBD with recombinant GTPases. a, saturation of binding. Recombinant Rac1 (20 pmol) was loaded with non-hydrolyzable [35S]GTPgS and incubated with increasing amount of GST-PBD, and then binding was analyzed as indicated under “Experi- mental Procedures.” Results shown are the mean 6 S.D. of 4–5 separate experiments. b, stability of binding. For competition binding experiments, 200 pmol of GST-PBD were incubated with 10 pmol of Rac1-[35S]GTPgS in the presence of increasing amounts of unlabeled Rac1-GTPgS. Unlabeled Rac1-GTPgS was either added simultaneously with the radiolabeled form (solid circles) or 30 (solid triangles) or 60 (open circles) min after prior incubation of GST-PBD with the labeled Rac1-[35S]GTPgS. After interaction for 30 min at 4 °C between Rac and GST-PBD, the affinity complex was precipitated with glutathione-Sepharose beads and washed, and the radioactivity bound to the bead pellet was counted. c, recombinant Cdc42 (50 ng) was loaded with [g-32P]GTP (solid lines) or [35S]GTPgS (dashed lines) and analyzed either in KRHG buffer or after addition to neutrophil cytosol, as indicated. Inhibition of the intrinsic GTP hydrolysis of Cdc42 by GST-PBD (solid lines) was measured by counting the amount of the [g-32P]GTP remaining associated with Cdc42 over a 20-min time course at 20 °C in the presence (open circles) or the absence (solid circles) of 4 mg of GST-PBD. Nucleotide dissociation was similarly assessed using [35S]GTPgS-loaded Cdc42 (dashed lines), and in the presence (open circle) or absence (solid circle) of GST-PBD. Values shown in b and c are the mean of three separate experiments.

Article Snippet: Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted for the appropriate GTPase using specific R786 (Rac2) and R785 (Rac1) antibodies (34), the Cdc42 polyclonal antibody from Santa Cruz Biotechnology (SC-87), and the RhoA monoclonal from Santa Cruz Biotechnology (SC-418).

Techniques: Binding Assay, Recombinant, Incubation, Labeling, Radioactivity, Inhibition

Journal: The Journal of biological chemistry

Article Title: Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.

doi: 10.1074/jbc.274.19.13198

Figure Lengend Snippet: FIG. 5. Rac2 and Cdc42 activation in HL-60 cells stimulated with fMLP is blocked by tyrosine kinase and PI 3-kinase inhib- itors. Me2SO-differentiated HL-60 cells suspended in KRHG/Ca21 (2 3 107 cells/ml) were treated 15 min at 37 °C in the presence of Me2SO (as control) or the following inhibitors: 100 mM genistein, 20 mM LY294002, or 30 nM wortmannin. Cells were then stimulated with 1 mM fMLP at 37 °C for 1 min, and activation was stopped by the addition of ice-cold 23 lysis buffer. Cell lysate was used for the affinity precipitation assay in the presence of 8 mg of GST-PBD. Proteins bound to GST-PBD were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted for Rac2 or Cdc42, followed by ECL detection. Values are the mean of three separate experiments; error bars represent standard deviation.

Article Snippet: Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted for the appropriate GTPase using specific R786 (Rac2) and R785 (Rac1) antibodies (34), the Cdc42 polyclonal antibody from Santa Cruz Biotechnology (SC-87), and the RhoA monoclonal from Santa Cruz Biotechnology (SC-418).

Techniques: Activation Assay, Inhibition, Control, Lysis, Affinity Precipitation, SDS Page, Membrane, Standard Deviation

Journal: Cell reports

Article Title: Functional Oocytes Derived from Granulosa Cells.

doi: 10.1016/j.celrep.2019.11.080

Figure Lengend Snippet: Figure 2. gPSCs Acquire High Germline Competence (A) Morphology and expression of pluripotency markers of gPSCs and female ESCs. Scale bar, 30 mm. (B) Low level of methylation at Oct4 and Nanog promoter regions in gPSCs. White dots indicate unmethylated loci, and dark spots indicate methylated loci. (C) Germline competency of gPSCs and female ESCs by injection into 4- to 8-cell embryos and genotyping by D12Mit136 microsatellite assay (right panel). Albino BALB/c mice served as embryo donors, and pseudo-pregnant albino Kunming mice served as surrogate mothers. (D) Summary of chimeras and germline transmission (GT) pups by injection into 4- to 8-cell embryos of gPSCs and female MEF-CiPSCs. (E) Scatterplots of genome-wide transcription. Parallel diagonal lines indicate the two-fold threshold in expression difference (p < 0.05).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Zscan4 Millipore Cat#: AB4340 Pan-Kcr PTM biolabs Cat#: PTM-502 Oct4 Santa Cruz Cat#: sc5279 b-Actin Abmart Cat#: P30002 Ki67 Millipore Cat#: AB9260 LaminA Abcam Cat#: ab26300 Foxl2 Abcam Cat#: ab5096 H3K27me3 Millipore Cat#: 07-499 Sox2 Millipore Cat#: AB5603 Nanog Abcam Cat#: ab80892 Lin28 CST Cat#: 3978S bIII-tubulin Chemicon Cat#: CBL412 a-SMA Abcam Cat#: ab5694 AFP Dako Cat#: DAK-N1501 Nestin Millipore Cat#: MAB353 a-Tubulin Sigma Cat#: SAB4800087 SCP1 Abcam Cat#: ab15090 SCP3 Novus Cat#: NB300-230 MLH1 BD Cat#: 550838 Texas Red-conjugated Phalloidin Abcam Cat#: ab176757 anti-SSEA1 conjugated with magnetic beads Miltenyi Cat#: 130-094-530 anti-SSEA1 conjugated with Alexa Fluor 647 eBioscience Cat#: 50-8813-42 anti-CD61 conjugated with PE BioLegend Cat#: 104307 FITC Goat Anti-Mouse IgG (H+L) Jackson Cat#: 115-095-003 Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L) Jackson Cat#: 111-585-003 Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Life Cat#: A11008 Alexa Fluor 594 Goat Anti-Mouse IgG (H+L) Life Cat#: A-11005 Alexa Fluor 594 Donkey Anti-Goat IgG (H+L) Abcam Cat#: ab150132 Goat anti-Rabbit IgG-HRP GE Healthcare Cat#: NA934V Goat anti-Mouse IgG (H+L)/HRP ZSGB-BIO Cat#: ZB2305 Bacterial and Virus Strains Trans1-T1 Phage Resistant Chemically Competent Cell Transgene Cat#: CD501-03 Chemicals, Peptides, and Recombinant Proteins Y27632 Selleck Cat#: S1049 PD0325901 Miltenyi Cat#: 04-0006 CHIR99021 Selleck Cat#: S1263 ESGRO (LIF) Millipore Cat#: ESG1107 Human bFGF PeproTech Cat#: 96-100-18B VPA Sigma Cat#: P4543 Repsox Selleck Cat#: S7223 Parnate Sigma Cat#: P8511 Forskolin Selleck Cat#: S2449 AM580 Tocris Cat#: 0760-10 (Continued on next page) e1 Cell Reports 29, 4256–4267.e1–e9, December 24, 2019

Techniques: Expressing, Methylation, Injection, Transmission Assay, Genome Wide