Journal: The Journal of biological chemistry

Article Title: Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 with cyclin A-Cdk2 and cyclin D2-Cdk4.

doi: 10.1074/jbc.272.41.25863

Figure Lengend Snippet: FIG. 1. Analysis of Cdk2- and p27-associated kinase activity in p27 inducible Mv1Lu cells. Tet-p27 cells were grown in media con- taining the indicated concentration of tetracycline for 18 h before they were assayed as follows. a, [125I]iododeoxyuridine incorporation into DNA at the end of the incubation (data are averages of triplicate determinations and are plotted as percent relative to the incorporation in the presence of 1 mg/ml tetracycline). b, immunoblotting of total cell extract (100 mg of protein) with p27 antibodies. c–e, p27 immunopre- cipitation followed by immunoblotting with anti-Cdk4 (c), anti-Cdk6 (d), or anti-Cdk2 (e). For reasons unknown, the recovery of p27-bound Cdk was low in extracts from cells incubated without tetracycline (data not shown). f and g, Cdk2 immunoprecipitation followed by histone H1 (f) or Rb (g) kinase assays. h, immunoblotting of total cell extract (200 mg of protein) with Rb antiserum. Rbphos indicates the hyperphospho- rylated form of Rb. i and j, p27 immunoprecipitation followed by histone H1 (i) or Rb (j) kinase assays. NRS, normal rabbit serum. Cdk2, Cdk2 immunoprecipitation, followed by kinase assay. k, left, in a separate experiment, Mv1Lu cell extracts were subjected to four cycles of immu- nodepletion with Cdk4 and Cdk6 antisera. These extracts were then immunoprecipitated with p27 antiserum (lane 5), followed by Cdk6 and Cdk4 immunoblotting. Lanes 1–4 are the immunocomplexes from each depletion round, analyzed by Cdk4 and Cdk6 immunoblotting. k, right, p27 was immunoprecipitated from lysates that had been subjected to four cycles of immunodepletion with Cdk4/Cdk6 or normal rabbit se- rum. The Rb kinase activity associated with the p27 immunocomplexes was then determined.

Article Snippet: Immunocomplexes were analyzed by Western immunoblot analysis with p27, Cdk4 (Pharmigen), or Cdk6 (Santa Cruz) antibodies by standard techniques.

Techniques: Activity Assay, Concentration Assay, Incubation, Western Blot, Immunoprecipitation, Kinase Assay, Immunodepletion

Journal: Cancer cell international

Article Title: MicroRNA-550a-3-5p controls the brain metastasis of lung cancer by directly targeting YAP1.

doi: 10.1186/s12935-021-02197-z

Figure Lengend Snippet: Fig. 7 The effects of miR-550a-3-5p on cleaved-PARP, pRB, CDK6, YAP1, CTGF, and CYR61. A Western blotting images. B Cleaved-PARP protein expression. C pRB protein expression. D CDK6 protein expression. E YAP1 protein expression. F CTGF protein expression. (G) CYR61 protein expression. *P < 0.05, compared with the control group; #P < 0.05, compared with the miR-550a-3-5p mimics group

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with anti-cleaved-Poly(ADP-ribose) polymerase (PARP) antibody (1:1000, Abcam), anti-RB transcriptional corepressor 1 (pRB) antibody (1:1000, Abcam), anti-cyclin dependent kinase 6 (CDK6) antibody (1:1000, Abcam), anti-Yes1 associated transcriptional regulator (YAP1) antibody (1:1000, Abcam), anti- connective tissue growth factor (CTGF) antibody (1:1000, Proteintech Group, Inc.), anti-cysteine rich angiogenic inducer 61 (CYR61) antibody (1:1000, Abcam), and anti-GAPDH antibody (1:10,000, Proteintech Group, Inc.) overnight at 4 °C.

Techniques: Western Blot, Expressing, Control

Journal: Oncology reports

Article Title: miR-3619-5p inhibits prostate cancer cell growth by activating CDKN1A expression.

doi: 10.3892/or.2016.5250

Figure Lengend Snippet: Figure 6. miR-3619-5p inhibits cell cycle related gene expression primarily by regulating CDKN1A expression. DU145 and PC3 cells were transfected with 50 nM of the indicated RNAs for 72 h. (A) Expression of p21 mRNA was assessed by real-time PCR. GAPDH served as a loading control. (B) Induction of p21 protein expression was detected by western blot analysis. GAPDH were also detected and served as a loading control. (C) Expression of cyclin D1, CDK4 and CDK6 mRNA was detected by real-time PCR. GAPDH served as a loading control. (D) Expression of cyclin D1, CDK4 and CDK6 mRNA was determined by western blot analysis. α-Tublin served as a loading control. *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: After blocking the membranes were incubated overnight at 4 ̊C with appropriate dilutions of specific primary antibodies as follows: p21 (1/2000) (Cell Signaling Technology), cyclin D1 (1/2000) (Affinity, USA), CDK4 (1/1000) (Affinity), CDK6 (1/2000) (Affinity), GAPDH (1/500) (Boster, Wuhan, China) and α-tubulin (1/500) (Boster).

Techniques: Gene Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot