Journal: Journal of Neuroscience Research

Article Title: The antiviral cytokine interferon‐gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation

doi: 10.1002/jnr.23987

Figure Lengend Snippet: IFNγ modulates the expression of cell cycle checkpoint proteins and the phosphorylation of pRb in NSPCs. A: Expression of cyclins D1, D2, D3, E, and cdk2 were measured using western blot and fluorescence signals were normalized to GAPDH as a loading control. For cyclin D1, the top band (open arrowhead) corresponds to the phosphorylated form of cyclin D1 and the bottom band (closed arrowhead) corresponds to the unphosphorylated form of cyclin D1. B: Expression of total retinoblastoma protein (pRb) and associated pRb phosphorylation at different serine residues (S780, S795, and S807/811) was measured. The fluorescence signal for each band was normalized to GAPDH as a loading control. For pRb S795, normalization was also performed against total pRb. Quantitation of samples is shown as the average with SEM. Statistical analysis was applied using repeated measures one‐way ANOVA with Bonferroni multiple comparisons post‐hoc analysis (****p < 0.0001, *** p < 0.001, **p < 0.01 *p < 0.5; n = 3‐5).

Article Snippet: Primary antibodies used were as follows: anti‐phospho STAT1 (Y701, #612133), anti‐STAT1 (N‐terminus, #610120), anti‐STAT3 (610190) from BD Biosciences; anti‐phospho STAT1 (S727, #9177) anti‐Cyclin D1 (#2978), anti‐Cyclin D3 (#2936); pRb S780 (#8180), pRb S807/811 (#8516), pRb S780 (#9307), Rb‐total (#9313) anti‐phospho STAT3 (#9131) form Cell Signaling Technology; pRb S795 (1:500, ab47474) from Abcam; and anti‐cdk4 (MAB8879), anti‐cdk2 (#07‐631) anti‐cyclin E (#07‐687) from Millipore.

Techniques: Expressing, Phospho-proteomics, Western Blot, Fluorescence, Control, Quantitation Assay

Journal: Journal of Neuroscience Research

Article Title: The antiviral cytokine interferon‐gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation

doi: 10.1002/jnr.23987

Figure Lengend Snippet: Characterization of Antibodies Used in Flow Cytometry and Western Blot Analyses.

Article Snippet: Primary antibodies used were as follows: anti‐phospho STAT1 (Y701, #612133), anti‐STAT1 (N‐terminus, #610120), anti‐STAT3 (610190) from BD Biosciences; anti‐phospho STAT1 (S727, #9177) anti‐Cyclin D1 (#2978), anti‐Cyclin D3 (#2936); pRb S780 (#8180), pRb S807/811 (#8516), pRb S780 (#9307), Rb‐total (#9313) anti‐phospho STAT3 (#9131) form Cell Signaling Technology; pRb S795 (1:500, ab47474) from Abcam; and anti‐cdk4 (MAB8879), anti‐cdk2 (#07‐631) anti‐cyclin E (#07‐687) from Millipore.

Techniques: Flow Cytometry, Western Blot, Molecular Weight, Concentration Assay, Recombinant, Infection, Expressing, Immunofluorescence, Virus, Protein-Protein interactions, Derivative Assay, Phospho-proteomics, Activation Assay, Activity Assay, Purification

Journal: Nature medicine

Article Title: Aurora kinase A drives the evolution of resistance to third generation EGFR inhibitors in lung cancer

doi: 10.1038/s41591-018-0264-7

Figure Lengend Snippet: a Immunoblot analysis of total and phosphorylated AURKA in PC9 and H1975 parental and acquired resistant cell lines treated with 1uM of the indicated inhibitors for 24 h. b Mean of cell proliferation of PC9 or H1975 cells transfected with plasmids expressing the indicated genes and treated 1uM EGFR-TKI for 72 h compared to DMSO treated cells performed in n=3 biologically independent samples. Significance based on comparison to LacZ control. c Immunoblot analysis 4 parental and 8 acquired resistant cell lines. Quantified intensities for pAURKA and TPX2 relative to the parental cell line is shown. d Proliferation compared to DMSO of PC9 and H1975 parental or acquired resistant cells treated with 1uM of osimertinib or rociletinib, 30nM of MLN8237 or the combination for 72 h. Mean over n=3 biologically independent samples. e Apoptosis measured by YO-PRO-1 positivity in the same models and drug treatments for 72 h. Shown is mean from n=3 biologically independent samples. f Mean tumor volume (mm 3 ) of PC9-RR xenografts during treatment with rociletinib (100mg/kg), MLN8237 (10mg/kg) or the combination. n=10 tumors in the vehicle and rociletinib arm, and n=7 in the MLN8237 and combination arm. g Percent change in tumor volume compared to baseline for individual PC9-OR cell xenografts teated for 11 days with osimertinib (5mg/kg), MLN8237 (10mg/kg) or the combination. P-value comparing combo to single agent osimertinib treatment. h Immunoblot of lysates from parental PC9, PC9-OR and PC9-RR cells treated with the indicated inhibitors or DMSO for 24 h. i Proposed mechanism for the efficacy of the combination in acquired resistant cells. P-values based two-tailed Student’s t-test. Error bars represent s.e.m. Blots are representative of at least two independent experiments. Full blots shown in .

Article Snippet: Antibodies for pEGFR (Y1068; 3777), pERK1/2 (T202/Y204; 4370), ERK1/2 (9102), pAKT (S473; 4060), AKT (9272), PARP (5625), pAURKA (T288; 3079), AURKA (4718) pRb (S780; 9307), BIM (2933), pBIM (S69; 4585), BAX (5023), Vimentin (5741), p-p65 (S536; 3033), p65 (8242), Pan phospho-AURKA/B/C (T288/T232/T198; 2914), CD44 (3570), Histone H3 (9715) were purchased from Cell Signaling Technology; TPX2 (HPA005487) and pan total AURKA/B/C (HPA002636) were purchased from Sigma; EGFR (SC-03), NEDD9 (sc-33657), AJUBA (sc-398008), PAK1 (sc-16617), CD24 (sc-19585), CD133 (sc-30219), B-tubulin (sc-9104), p53 (sc-126) were purchased from Santa Cruz biotechnology and FZR1/CDH1 (ab3242) from Abcam and V5 tag (46–0705) from Thermofisher.

Techniques: Western Blot, Transfection, Expressing, Comparison, Control, Two Tailed Test

Journal:

Article Title: Cell Cycle Inhibition by FoxO Forkhead Transcription Factors Involves Downregulation of Cyclin D

doi: 10.1128/MCB.22.22.7842-7852.2002

Figure Lengend Snippet: FoxO factor expression results in reduced protein levels of cyclins D1 and D2 and inhibits activity of cyclin D/CDK4 complexes in vivo. (A) NIH 3T3 cells were transiently transfected with an expression vector for Myc-tagged pRb in combination with either an empty control plasmid, a vector encoding full-length HA-FoxO4, or the indicated FoxO4 mutants, respectively. Forty-eight hours after transfection, exogenous Myc-pRb was immunoprecipitated from total lysates by using a monoclonal anti-Myc antibody, and CDK4-mediated phosphorylation of immunoprecipitated Myc-pRb was analyzed by Western blotting with a phosphospecific antibody against S780 (22). Similar loading of immunoprecipitated Myc-pRb was confirmed by staining the same blot with antiserum against total pRb. Phosphorylated pRb (ppRb) (upper band) migrates more slowly than unphosphorylated pRb (pRb) (lower band). (B) MEFs from wild-type (p27+/+) or p27kip1-deficient (p27−/−) embryos were infected with an HA-FoxO4-containing retrovirus (FoxO4) or a control retrovirus (puro). Forty-eight hours later, cells were harvested and total lysates were analyzed by Western blotting for the presence of HA-FoxO4, cyclin D1 (Cyc D1), or p27kip1. The cyclin D1 blot was additionally immunostained for CDK4 as a loading control. The activity of cyclin D/CDK4 complexes was judged by immunoblotting for phospho-S780pRb (pS780pRb). (C) Immortalized p27kip1−/− MEFs were infected as described for panel B and analyzed for protein expression of cyclin D1 (Cyc D1), cyclin D2 (Cyc D2), and HA-FoxO4 by Western blotting. CDK4 served as a loading control.

Article Snippet: Anti-cyclin E (sc-481), anti-CDK4 (sc-270), anti-phospho S780 pRb (sc-12901), and anti-cyclin D2 (sc-452) were purchased from Santa Cruz.

Techniques: Expressing, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Infection

Journal:

Article Title: Cell Cycle Inhibition by FoxO Forkhead Transcription Factors Involves Downregulation of Cyclin D

doi: 10.1128/MCB.22.22.7842-7852.2002

Figure Lengend Snippet: Conditional activation of FoxO3a results in downregulation of cyclin D1 and D2 proteins and inhibition of CDK4 activity in human colon carcinoma cells. DL23 and DLD-1 cells were left untreated (0 h) or treated with 100 nM 4-OHT for the indicated time periods. Total lysates were prepared and subsequently analyzed by Western blotting for the presence of cyclin D1 (Cyc D1) (A), or cyclin D2 (Cyc D2) and p27kip1 (B, lower panel). Additionally, total phosphorylation of pRb (ppRb) (B, upper panel) or of phospho-pRb S780 (pS780pRb) (B, lower panel) was determined by using an antibody against total pRb or a phosphospecific antiserum against S780 of the pRb protein, respectively. Equal loading for panels A and B was confirmed by Western blotting for CDK4.

Article Snippet: Anti-cyclin E (sc-481), anti-CDK4 (sc-270), anti-phospho S780 pRb (sc-12901), and anti-cyclin D2 (sc-452) were purchased from Santa Cruz.

Techniques: Activation Assay, Inhibition, Activity Assay, Western Blot

Journal:

Article Title: Commitment Point during G 0 ->G 1 That Controls Entry into the Cell Cycle

doi: 10.1128/MCB.23.7.2351-2361.2003

Figure Lengend Snippet: Identifying a G0→G1 commitment point in nonactivated human T cells stimulated with CD3/CD28. (A) Samples were taken at the times shown after CD3/CD28 stimulation, and the cellular DNA (PI) and protein (FITC) content were determined by flow cytometry. Cell size and protein content increase as cells enter and traverse G1, and the percentages in the G0, G1, and S+G2/M phases are shown. (B) The kinetics of pRb phosphorylation and the induction of proteins encoded by several E2F-regulated genes during cell cycle entry were determined by Western blotting, with samples from the same experiment as shown in panel A. Antibodies used recognize phosphorylation at the cdk6/4-cyclin D-specific sites S780 and S807/811, as well as a site phosphorylated by cdk2-cyclin E, T821. Proteins of <30 kDa (mainly histones) were stained with Coomassie blue as a loading control. (C and D) In vitro kinase assays. cdk6, cdk4, and cdk2 were immunoprecipitated at the times shown after CD3/CD28 stimulation, and their activities were assayed with recombinant pRb as the substrate. The 32P-labeled pRb was separated by gel electrophoresis, transferred to a nitrocellulose membrane, and detected by exposure to film. The assays were carried out with three independent T-cell preparations, and each assay was consistent with the data shown in panel C. Complete time courses were available for two of the experiments, and these are quantified in panel D (mean ± range). After autoradiography, the membranes were subjected to Western blotting for cyclin D2, D3, and E in order to detect the association of these cyclins with the relevant cdk at each time point (C). (E and F) The CD4+ and CD8+ T-cell subsets were stimulated transiently with CD3/CD28 for the times shown. Samples were obtained at 48 h, and the cellular DNA and protein content (E), as well as the pRb phosphorylation and E2F-dependent protein expression (F), were evaluated. In panel F, blots were probed for ERK as a loading control. In panel E, data for CD8 cells are shown with the percentages in the G0, G1, and S+G2/M phases for both the CD4 and the CD8 subsets. The data are representative of three separate experiments.

Article Snippet: The antibodies used here were Mcm2 and Mcm5 ( 49 , 50 ); Mcm2 (BM28; Transduction Laboratories, Becton Dickinson); Mcm3 (N19), cdc2 ( 17 ), Cdc6 (C19), cdk4 (H22), cdk6 (C21), c-myc (9E10), p130 (C20), p107 (C18), cyclin D2 (C17), and cyclin D3 (C16) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); cyclin D2 (Ab4; DCS3.1 and DCS5.2; Neomarker, Fremont, Calif.); phospho-pRb (S 780 and S 807/811 ; New England BioLabs, Hertfordshire, United Kingdom) (T 821 ; Biosource, Camarillo, Calif.); or pRb (PMG3245; Becton Dickinson).

Techniques: Flow Cytometry, Western Blot, Staining, In Vitro, Immunoprecipitation, Recombinant, Labeling, Nucleic Acid Electrophoresis, Autoradiography, Expressing

Journal:

Article Title: Commitment Point during G 0 ->G 1 That Controls Entry into the Cell Cycle

doi: 10.1128/MCB.23.7.2351-2361.2003

Figure Lengend Snippet: pTAT-p16INK4A and roscovitine inhibit the pRb/p130 pathways and prevent transition through the G0→G1 commitment point. (A to C) Nonactivated T cells were transduced with TAT-p16INK4A or TAT-p16MUT (inactive mutant of p16INK4A), or roscovitine was added at the concentrations shown. The cells were stimulated with CD3/CD28 (A) or PMA-ionomycin (B and C), and samples were taken at 24 and 48 h. The pRb phosphorylated on S807/811, the total pRb and p130 proteins, and the E2F-regulated Cdc6, cdc2 p107, and cyclin D3 proteins were analyzed by Western blotting. pRb, total pRb protein; 3, hyperphosphorylated p130; 1,2, hypophosphorylated p130. n/s, not stimulated with CD3/CD28 or PMA-ionomycin. n/t, not transduced with TAT-p16INK4A or TAT-p16MUT. Histones stained with Coomassie blue were a loading control for panel C. (D) Inhibition of cdk activity by roscovitine. T cells were stimulated with CD3/CD28 in the presence (+) and absence (−) of 20 μM roscovitine, and the activity of each cdk was determined by in vitro kinase assays. (E) Western blot of phospho-IκB (S32) and ERK1/2 phosphorylated at T202/Y204. Blots of total cell IκB and ERK1/2 proteins are also shown. (F) Western blot of c-myc 5 h after stimulation. n/s, not stimulated; n/t, stimulated but not transduced with TAT protein. (G) Inhibition of cdk activation prevents the formation of DNA origin recognition complexes. T cells were transduced with TAT-p16INK4A or TAT-GFP (as a control), and extracts of chromatin-bound and free proteins were prepared at the times shown. The presence of Mcm2, Mcm3, and Cdc6 in each sample was determined by Western blotting. Histones and cdk6 are loading controls for chromatin-bound and free proteins, respectively. (H) The T cells were stimulated with PMA-ionomycin and transduced with TAT-p16INK4A or TAT-p16MUT at 0, 1, or 5 h after stimulation. Samples were obtained at 18, 24, and 30 h for the analysis of pRb phosphorylation and chromatin binding of Mcm2. cdk6 was used as a loading control for total cell lysates and for extracts of non-chromatin-bound (free) protein and histones stained with Coomassie blue for chromatin. The data shown in panels B, C, and G are each representative of six separate experiments; the data shown in panels A, E, F, and H are each representative of three experiments, and the data shown in panel D are representative of two experiments.

Article Snippet: The antibodies used here were Mcm2 and Mcm5 ( 49 , 50 ); Mcm2 (BM28; Transduction Laboratories, Becton Dickinson); Mcm3 (N19), cdc2 ( 17 ), Cdc6 (C19), cdk4 (H22), cdk6 (C21), c-myc (9E10), p130 (C20), p107 (C18), cyclin D2 (C17), and cyclin D3 (C16) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); cyclin D2 (Ab4; DCS3.1 and DCS5.2; Neomarker, Fremont, Calif.); phospho-pRb (S 780 and S 807/811 ; New England BioLabs, Hertfordshire, United Kingdom) (T 821 ; Biosource, Camarillo, Calif.); or pRb (PMG3245; Becton Dickinson).

Techniques: Transduction, Mutagenesis, Western Blot, Staining, Inhibition, Activity Assay, In Vitro, Activation Assay, Binding Assay