kpna3 antibody  (Bio-Techne corporation)

Journal: Molecular Cancer

Article Title: Antitumor effects of pharmacological EZH2 inhibition on malignant peripheral nerve sheath tumor through the miR-30a and KPNB1 pathway

doi: 10.1186/s12943-015-0325-1

Figure Lengend Snippet: DZNep inhibited EZH2/miR-30d/KPNB1 signaling in MPNST cells. (A) Western blot analyses of EZH2, cleaved PARP (cPARP), KPNB1, and GAPDH (loading control) in MPNST724 and S462 cells treated with DZNep at increasing concentrations for 48 hours. (B) qRT-PCR analyses of miR-30d expression in MPNST724 and S462 cells treated with DZNep for 96 hours. miR-30d expression was normalized to SNORD47. Mean ± SD values are shown (n = 3); *p < 0.05, Student t test. (C) Promoter activity assay showed that DZNep treatment increased miR-30d promoter activity in S462 cells. Mean ± SD values are shown (n = 3); *p < 0.05; Student t test. (D) Luciferase reporter assay showed that DZNep treatment inhibited miR-30d target reporter activity in S462 cells. Mean ± SD values are shown (n = 3); **p < 0.01, Student t test. (E) Luciferase reporter assay indicated that DZNep treatment inhibited wild-type (WT) KPNB1 3’UTR reporter activity but not mutant (MT) KPNB1 3’UTR reporter activity in S462 cells. Mean ± SD values are shown (n = 3); *p < 0.05, Student t test.

Article Snippet: Commercially available antibodies were used for all immunoblot and immunohistochemical detection of EZH2 (1:1000, D2C9, Cell Signaling ) , KPNB1 (1:5000, NB100-81650, Novus Biologicals,), cleaved PARP (1:1000, ab32064, Abcam), vimentin (1:2000, RV202, Santa Cruz), E-cadherin (1:1000, H-108, Santa Cruz), Ki-67 (1:1000, MIB-1; Dako), cleaved caspase 3 (1:1000, BioCare Medical), GAPDH-HRP (1:5000, ab9483, Abcam), and actin-HRP (1:5000, I-19, Santa Cruz).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Activity Assay, Luciferase, Reporter Assay, Mutagenesis

Journal: Molecular Cancer

Article Title: Antitumor effects of pharmacological EZH2 inhibition on malignant peripheral nerve sheath tumor through the miR-30a and KPNB1 pathway

doi: 10.1186/s12943-015-0325-1

Figure Lengend Snippet: DZNep suppressed MPNST724 xenograft tumor initiation and growth in vivo . (A) MPNST724 xenograft tumor groups treated with vehicle only, 1 mg/kg DZNep, or 5 mg/kg DZNep 13 weeks after subcutaneous injection (n = 9 mice per group). (B) Growth curves of tumors treated with vehicle only, 1 mg/kg DZNep, or 5 mg/kg DZNep over a period of 13 weeks. Mean ± SD values are shown; *p < 0.05, Student t test. (C) Western blot analyses of EZH2, KPNB1, and actin in tumor MPNST samples treated with vehicle only, 1 mg/kg DZNep, or 5 mg/kg DZNep.

Article Snippet: Commercially available antibodies were used for all immunoblot and immunohistochemical detection of EZH2 (1:1000, D2C9, Cell Signaling ) , KPNB1 (1:5000, NB100-81650, Novus Biologicals,), cleaved PARP (1:1000, ab32064, Abcam), vimentin (1:2000, RV202, Santa Cruz), E-cadherin (1:1000, H-108, Santa Cruz), Ki-67 (1:1000, MIB-1; Dako), cleaved caspase 3 (1:1000, BioCare Medical), GAPDH-HRP (1:5000, ab9483, Abcam), and actin-HRP (1:5000, I-19, Santa Cruz).

Techniques: In Vivo, Injection, Western Blot

Journal: Molecular Cancer

Article Title: Antitumor effects of pharmacological EZH2 inhibition on malignant peripheral nerve sheath tumor through the miR-30a and KPNB1 pathway

doi: 10.1186/s12943-015-0325-1

Figure Lengend Snippet: EZH2-regulated miR-30a targeted KPNB1 in MPNST cells. (A) Putative miR-30a and miR-30d target site in the wild-type KPNB1 3’UTR region. (B) qRT-PCR analyses of miR-30a in MPNST724 and S462 cells transfected with a negative control or EZH2 siRNA. miR-30a expression was normalized to SNORD47. Data are shown as mean ± SD (n = 3); *p < 0.05, Student t test. (C) qRT-PCR analyses of miR-30a in S462 and MPNST724 cells treated with DZNep for 96 hours. miR-30d expression was normalized to SNORD47. Mean ± SD values are shown (n = 3); *p < 0.05, Student t test. (D) Western blot analyses of KPNB1 and actin in S462 and MPNST724 cells transfected with a negative control (Ctl) or miR-30a (30a) mimics. (E) Luciferase reporter assay showed that miR-30a inhibited wild-type (WT) KPNB1 3’UTR reporter activity but not mutant (MT) KPNB1 3’UTR reporter activity in S462 cells. Mean ± SD values are shown (n = 3); *p < 0.05, Student t test. (F) qRT-PCR analyses of miR-30a expression in normal Schwann cells (NSC) and multiple MPNST cell lines (MPNST724, S462, STS26T, MPNST624, T265, and ST88-14).

Article Snippet: Commercially available antibodies were used for all immunoblot and immunohistochemical detection of EZH2 (1:1000, D2C9, Cell Signaling ) , KPNB1 (1:5000, NB100-81650, Novus Biologicals,), cleaved PARP (1:1000, ab32064, Abcam), vimentin (1:2000, RV202, Santa Cruz), E-cadherin (1:1000, H-108, Santa Cruz), Ki-67 (1:1000, MIB-1; Dako), cleaved caspase 3 (1:1000, BioCare Medical), GAPDH-HRP (1:5000, ab9483, Abcam), and actin-HRP (1:5000, I-19, Santa Cruz).

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Expressing, Western Blot, Luciferase, Reporter Assay, Activity Assay, Mutagenesis