anti-ssar  (Novus Biologicals)

Journal: Acta Neuropathologica

Article Title: A driver role for GABA metabolism in controlling stem and proliferative cell state through GHB production in glioma

doi: 10.1007/s00401-016-1659-5

Figure Lengend Snippet: Loss of stem and tumorigenic properties by GBM stem-like cells is accompanied with GABA metabolism deregulation characterized by enhanced GHB levels. a Increased GABA by-products to α-KG ratios in TG1-miR compared to TG1. The “+” sign represents the mean value in the whisker box . Mean ± SD, n = 6 independent biological samples. b Schematic reconstruction of metabolic pathways with green and red boxes signaling metabolites decreased or increased in TG1-miR compared to TG1, respectively. Enzyme names are within grey boxes . When relevant, the corresponding gene designation is indicated below the enzyme name. SSADH: succinic semialdehyde dehydrogenase. SSAR succinic semialdehyde reductase. c Downregulation of the ALDH5A1 protein product SSADH in TG1-miR. Western blot analysis. SSADH MW, 57 kDa; Actin MW, 42 kDa. Mean ± SD, n = 3 independent biological samples. d Decreased ALDH5A1 mRNA levels in TG1-miR compared to TG1. Q-PCR assays. Mean ± SD, n = 3 independent biological samples. e Targeting of the ALDH5A1 transcript by miR-302. Expression of Renilla Luciferase mRNA containing the wild-type form of ALDH5A1 -3′UTR is strongly reduced in TG1-miR compared to TG1. Deletion of miR-302 putative target sequence in the 3′UTR of ALDH5A1 mRNA ( ALDH5A1 -3′UTR-DEL) prevents the binding of the miR, and rescues luciferase activity. n = 3 independent biological samples. f Decreased SSADH levels in GBM and DIPG stem-like cells (TG1, TP54) upon ALDH5A1 downregulation with siRNAs (siA). siC (control siRNA). Mean ± SD, n = 3 independent biological samples. RDU, relative densitometry units. g ALDH5A1 down regulation results in enhanced GHB intra-cellular levels. Mean ± SD, n = 3 independent biological samples. h SSADH immunoreactive cells are enriched in proliferative/non-differentiated GBM territories ( P HIGH / D − ) and rare in non-proliferative/differentiated ( P LOW / D + ) tumor territories of patients’ GBM, as revealed by immunohistochemical staining of Ki67, Olig2, and GFAP. HES: hematoxylin and eosin staining. Scale bar 100 µm. i GHB/α-KG ratios in proliferative/non-differentiated ( P HIGH / D − ) and weakly proliferative/differentiated territories ( P LOW / D + ) of patient GBM. GC–MS/MS analysis. Mean ± SD, n = 5 independent patient’s GBM neurosurgical samples

Article Snippet: The following antibodies were used for immunoblotting: anti-Nanog (Cell Signaling, 1:1000), anti-p21 (Santa Cruz Biotechnology, 1:200), anti-Actin (Millipore Chemicon, 1:10,000), anti-SSAR (Novus biological, 1:2000), anti-ABAT1 (GABA-T) (Sigma Aldrich, 1:2500), anti-GAD65 (Abcam, 1:500), anti-GAD67 (Abcam, 1:500), anti-HOT (Sigma, 1:2000), anti-GLUD1 (Sigma, 1:2000), and anti-SSADH (Novus Biological, 1:2000).

Techniques: Whisker Assay, Western Blot, Expressing, Luciferase, Sequencing, Binding Assay, Activity Assay, Immunohistochemical staining, Staining, Gas Chromatography-Mass Spectrometry