primary monoclonal rabbit anti-stathmin antibody  (Spring Bioscience)

Journal: Proteome Science

Article Title: Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma

doi: 10.1186/s12953-016-0094-9

Figure Lengend Snippet: Proteomics analysis of differentially expressed proteins in U87MG transfected with siRNA for MELK compared with U87MG transfected with non-target control. a Representative two-dimensional electrophoresis gels (2DE). Protein extracts (200 μg) were applied to 2DE using IPG pH 3-10NL (7 cm) for isoelectric focusing and 12.5 % SDS-PAGE mini gels as a second dimension. Gel image analysis was performed using ImageMaster 2D Platinum v.7.0 software. The arrows indicate selected protein spots from mass spectrometry analysis. b Proteins differentially expressed (up-regulated and downregulated) in the glioma cell line U87MG transfected with siRNA for MELK compared with U87MG cells transfected with non-target control (NTC). The expression levels of 12 differentially expressed protein spots were quantified on the basis of the normalised volume of the 2DE spots (% vol) for each group. These data were analysed via ANOVA (p < 0.05). The data are reported as the mean ± SD. One of the proteins, Stathmin (STMN1), exhibited lower expression levels when MELK was silenced

Article Snippet: The membrane was blocked with 5 % skim milk and incubated with primary polyclonal rabbit anti-MELK antibody (1:2000, Sigma-Aldrich) and primary monoclonal rabbit anti-Stathmin antibody (1:1000, Spring Bioscience, clone SP49, Pleasanton, CA).

Techniques: Transfection, Electrophoresis, SDS Page, Software, Mass Spectrometry, Expressing

Journal: Proteome Science

Article Title: Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma

doi: 10.1186/s12953-016-0094-9

Figure Lengend Snippet: MELK and stathmin protein expression in non-neoplastic and astrocytoma tissue samples. Immunohistochemistry was performed for MELK a – f and stathmin ( g – l ) in 6 cases each of in ( a and g ) non-neoplastic (NN) brain tissues, ( b and h ) pilocytic (AGI), ( c and i ) low grade (AGII), ( d and j ) anaplastic (AGIII) astrocytomas, and ( e and k ) glioblastoma (GBM). Pictures show representative cases of each sample type, with 400-x magnification. The bar ( k ) represent the scale bar of 10 μm. The graphs ( f and l ) represent a semi-quantitative immunolabelling score (ILS) calculated as the product of staining intensity and the percentage of positive cells. Both MELK and stathmin showed positive staining in the cytosol. MELK reactivity increased with the increment of the malignancy among diffusely infiltrating astrocytomas (AGII-AGIV). Stathmin cytosol staining was stronger in GBM cases. The horizontal bars show the median ILS of each group

Article Snippet: The membrane was blocked with 5 % skim milk and incubated with primary polyclonal rabbit anti-MELK antibody (1:2000, Sigma-Aldrich) and primary monoclonal rabbit anti-Stathmin antibody (1:1000, Spring Bioscience, clone SP49, Pleasanton, CA).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Proteome Science

Article Title: Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma

doi: 10.1186/s12953-016-0094-9

Figure Lengend Snippet: Effect of MELK and STMN1 knockdown with siRNA in GBM cell line. MELK and STMN1 expression levels relative to non-targeting control (NTC) were analyzed two days after transfection of U87MG cell line a The data show the average of two independent experiments and the vertical bar represents the standard deviations. Western blot of MELK and stathmin were analyzed after trasfection with siRNA for both genes and NTC ( b )

Article Snippet: The membrane was blocked with 5 % skim milk and incubated with primary polyclonal rabbit anti-MELK antibody (1:2000, Sigma-Aldrich) and primary monoclonal rabbit anti-Stathmin antibody (1:1000, Spring Bioscience, clone SP49, Pleasanton, CA).

Techniques: Expressing, Transfection, Western Blot

Journal: Proteome Science

Article Title: Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma

doi: 10.1186/s12953-016-0094-9

Figure Lengend Snippet: MELK and stathmin signaling. MELK can induce stathmin expression through two transcription factors, p53 and FOXM1. A tyrosine kinase receptor (TKR) is activated by phosphorylation after binding to a ligant. Growth factor receptor-bound protein 2 (GRB2) binds to the phophorylated residue of TKR and to Son of Sevenless homologs (SOS). GRB/SOS complex activates phosphoinositide 3-kinase (PI3K) and RAS-MAPK signaling pathways. Activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways results in phosphorylation of stathmin on serine sites and consequent microtubule stability and cell cycle progression. Stathmin may be dephosphorylated by protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B), resulting in microtubule instability. TKR: tyrosine kinase receptor; P: phosphorylation

Article Snippet: The membrane was blocked with 5 % skim milk and incubated with primary polyclonal rabbit anti-MELK antibody (1:2000, Sigma-Aldrich) and primary monoclonal rabbit anti-Stathmin antibody (1:1000, Spring Bioscience, clone SP49, Pleasanton, CA).

Techniques: Expressing, Binding Assay, Activation Assay

Journal: Proteome Science

Article Title: Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma

doi: 10.1186/s12953-016-0094-9

Figure Lengend Snippet: Protein identification by MALDI-TOF-TOF MS of tryptic peptides obtained from 2DE spots of siRNA-NTC- and siRNA- MELK -transfected cells

Article Snippet: The membrane was blocked with 5 % skim milk and incubated with primary polyclonal rabbit anti-MELK antibody (1:2000, Sigma-Aldrich) and primary monoclonal rabbit anti-Stathmin antibody (1:1000, Spring Bioscience, clone SP49, Pleasanton, CA).

Techniques: Transfection