rabbit polyclonal anti-human cyclin d2  (Santa Cruz Biotechnology)

Journal: Molecular and cellular endocrinology

Article Title: DUAL INHIBITION OF ERK1/2 AND AKT PATHWAYS IS REQUIRED TO SUPPRESS THE GROWTH AND SURVIVAL OF ENDOMETRIOTIC CELLS AND LESIONS

doi: 10.1016/j.mce.2018.12.011

Figure Lengend Snippet: Details of the antibody used.

Article Snippet: Anti-human rabbit polyclonal Cyclin D2 , Cell Signaling , 2924 , 1:1000.

Techniques: Concentration Assay

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 2. Analysis of Downstream Signaling of H-RasV12 in GSCs (A) Real-time PCR analysis of H-RasV12-GSCs (n = 3). Cells were cultured on laminin for 6 days under the indicated conditions. The values were normal- ized to Hprt1 expression, with expression levels in WT GSCs cultured with cytokines. (B) The patterns of cyclin expression during culture on laminin. The values were normalized to Hprt1 expression, with expression levels of cyclin D1. (C) Induction of cyclin expression in WT GSCs on laminin. Cells were stimu- lated with the indicated cytokines for 24 hr. The values were normalized to Hprt1 expression, with expression levels in cells cultured without cytokines. (D) Western blot analysis of WT and H-RasV12-GSCs. The cells were starved on laminin for 4 days, and then WT GSCs were either treated or not treated with the indicated cytokines. Treated cells were recovered 30 min after treatment. E, EGF; F, bFGF; G, GDNF. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Western Blot

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 3. GSC Proliferation by Cyclin Trans- fection (A) RT-PCR analysis of cyclin expression in cyclin D-transfected cells. The cells were cultured on laminin for 24 hr under the indicated conditions. (B) Effect of cytokines on cyclin-transfected GSC growth. WT GSCs were transfected with cyclin D genes. The cells were cultured under the indicated conditions on MEFs for 6 days. (C) Increased expression of cyclin E after addi- tional cyclin E transfection (n = 6). The cells were cultured on laminin for 24 hr without cytokines. Values were normalized to Hprt1 expression, with expression levels in WT GSCs. (D) Appearance of cyclin-transfected cells cultured without cytokines on MEFs for 6 days. Only cyD2E-GSCs formed germ cell colonies. (E) Effect of cytokines on GSCs that were trans- fected with cyclin D and E. While H-RasV12- GSCs and cyD2E-GSCs grew without cytokines, cycD1E-GSCs and cyD3E-GSCs grew when they were supplemented with EGF and bFGF. The cells were cultured on MEFs for 6 days. (F and G) Real-time PCR analysis of cyclin-trans- fected GSCs (n = 6). Cells were cultured with (F) or without (G) cytokines on laminin for 24 hr. Values were normalized to Hprt1 expression, with expression levels in WT GSCs. Scale bar, 100 mm (D). E, EGF; F, bFGF; G, GDNF. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 4. Phenotypic Characterization of H-RasV12-GSCs and cyclin-Transfected Cells (A) Cell-cycle distribution. Significantly more cells are in the G2/M phase in the H-RasV12-GSCs and cyD2E-GSCs. (B) Characterization of cell surface antigens by flow cytometry. Note the weaker expression of EpCAM and a6- and b1-integrin in cyD1E-GSCs and cyD3E-GSCs. Red line, specific antibody; black line, unstained control. Values indicate mean fluorescence intensity. (C) Statistically significant reduction in laminin binding of cyD1E- and cyD3E-GSCs. (D) RT-PCR analysis. Neurog3 expression was weaker in both cyD2E- and cyD3E-GSCs. (E) Immunocytochemistry of p27 and cyclin D2 in transfected cells. The transfectants were cultured on laminin without cytokines for 6 days and stained with anti-cyclin D2 (top) or p27 (bottom) antibody. Cyclin D2 was strongly expressed in both WT and H-RasV12-GSCs. p27 staining was predominantly found in the cytoplasm of H-RasV12-GSCs and cyD2E-GSCs, whereas nuclear staining was found in cyD1E-GSCs and D3E-GSCs. Counterstained by DAPI. (F) COBRA. Open arrows indicate the sizes of the methylated DNA, whereas closed arrows indicate the size of the unmethylated DNA. Percent methylation, as estimated by the intensity of each band, is indicated below the gels. U, uncleaved; C, cleaved. Scale bar, 10 mm (E). E, EGF; F, bFGF; G, GDNF. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Transfection, Cytometry, Expressing, Control, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry, Cell Culture, Staining, Combined Bisulfite Restriction Analysis Assay, Methylation

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 6. A Model for SSC Self-Renewal Growth signals are converted to Ras activation via Src family molecules. Ras transmits signals to activate the PI3K-Akt pathway as well as other unknown pathways that run parallel to it. Cyclins D2 and E coordinate to drive SSC self-renewing division by eliminating p27 from the nucleus and upregulating b1-integrin, whereas strong cyclin D1 expression may induce differentiation.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Activation Assay, Expressing