human il20 elisa kit  (PeproTech)

Journal: Oncotarget

Article Title: Ell3 stabilizes p53 following CDDP treatment via its effects on ubiquitin-dependent and -independent proteasomal degradation pathways in breast cancer cells

doi:

Figure Lengend Snippet: A. To confirm ERK phosphorylation, control (C) and Ell3-overexpressing (OE) MCF7 cells were pretreated with the ERK inhibitor PD980589 (50 μM) or DMSO vehicle for 24 h and p53 levels were analyzed by western blotting 16 h after CDDP treatment. B. The effect of PD98059 on apoptosis of control and Ell3-OE cells was quantified by annexin V staining and flow cytometry at 48 h after CDDP treatment. C. The five most up-regulated genes in ELL-OE cells, compared with control cells, determined by microarray analysis. D. The effect of si IL20 on ERK phosphorylation and p53 stabilization in Ell3-OE cells after CDDP treatment. Non-specific siRNA (siNS) or siRNA targeting IL20 (si IL20 ) was transfected into control and Ell3-OE cells for 24 h. Total p53 and phospho-ERK were analyzed by western blotting at 16 h after CDDP treatment. E. The effect of si IL20 on the apoptosis of control and Ell3-OE cells following CDDP treatment for 48 h was quantitatively measured by flow cytometry. F. The effect of anti-IL20 antibodies added to the culture media of Ell3-OE on CDDP-induced p53 protein accumulation. To neutralize IL20, 4 μg IL20 antibody was added to the medium containing 6 × 10 4 cells for 4 h, and the p53 protein level was analyzed by western blotting at 4 h after CDDP treatment with culture medium containing 1% FBS. G. The effect of si Ell3 on the expression of IL20 in Ell3-OE cells was analyzed by real-time PCR. Total RNA was analyzed 48 h after siRNA transfection. H. The effect of si Ell3 and si IL20 on the amount of secreted IL20 from Ell3-OE cells. After 24 h of siRNA transfection, IL20 in the culture media of control and Ell3-OE cells was measured using an IL20 immunoassay kit. I. Chromatin immunoprecipitation analysis of the transcription initiation region of the IL20 promoter in control and Ell3-OE using antibodies against IgG (negative control), Ell3, and acetylated histone H3 (Ace-H3). Binding of Ell3 to the promoter of IL20 was quantified by PCR and real-time PCR. The error bars represent the SE from three independent experiments, each performed with triplicate samples. * P < 0.05, ** P < 0.01, Student's t -test.

Article Snippet: A human IL20 immunoassay kit was used (DL200, R&D Systems).

Techniques: Phospho-proteomics, Control, Western Blot, Staining, Flow Cytometry, Microarray, Transfection, Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Binding Assay

Journal: Oncotarget

Article Title: Ell3 stabilizes p53 following CDDP treatment via its effects on ubiquitin-dependent and -independent proteasomal degradation pathways in breast cancer cells

doi:

Figure Lengend Snippet: Ell3 expression leads to the suppression of MDM2 expression and activation of the IL20 mediated ERK1/2 signaling pathway. Due to the low level of MDM2, p53 is not ubiquitinated following CDDP treatment. Additional ERK1/2 is phosphorylated following CDDP treatment, resulting in increased binding affinity of NQO1 to p53. The synergistic effect of suppressing the MDM2 pathway and enhancing the IL20 pathway causes rapid stabilization of p53 in Ell3-overexpressing breast cancer cells following CDDP treatment.

Article Snippet: A human IL20 immunoassay kit was used (DL200, R&D Systems).

Techniques: Expressing, Activation Assay, Binding Assay