Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: (A) Schematic representation of GAT-1 protein topology and locations of GAT-1 variants identified in patients associated with a spectrum of epilepsy syndromes. It is predicted that GAT-1 contains 12 transmembrane domains. G234S is located at the junction of the second intracellular loop and the 5th transmembrane domain of the GAT-1 protein. The positions of variants are based on the published LeuT crystal structure. (B) Amino acid sequence homology shows that glycine (G) at residue 234 is highly conserved in SCL6A1 in human (Accession NO. NP_003033.3) and across species. (C) Tertiary structures of both the wildtype and G234S mutant protein GAT-1 are predicted by I-TASSER. Residue 234 is highlighted as red and Glycine is mutated to Serine. (D) From interatomic interactions predictions by DynaMut, wild-type (upper) and G234S mutation (bottom) residues are colored in light-green and are also represented as sticks alongside with the surrounding residues. Halogen bonds are depicted in blue. Hydrogen bonds are colored in red. Machine learning methods as Supplementary Table 1 predicted this mutation destabilized the global conformation of the GAT-1 protein.

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Sequencing, Mutagenesis

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: A-B. HEK293T cells were transfected with GAT-1YFP (3μg) for 48 hrs. (A) Total lysates were analyzed by SDS-PAGE and western blot. The membranes were blotted with mouse anti-GFP antibody. (B) Rat cortical neurons were transfected with the wildtype or the mutant GAT-1(G234S)YFP cDNAs at day 7 days old in cultured dish. The total lysates were harvested from rat cortical neurons expressing the wildtype GAT-1YFP (wt) or mutant GAT-1(G234S)YFP (G234S) transporters after 8 days of transfection. The total lysates were then analyzed by SDS-PAGE. In HEK 293T cells (A), three protein bands were detected in both the wildtype and the mutant conditions. In rat cortical neurons (B), only a single strong band was detected in both the wildtype and the mutant conditions. In A and B, 1:500 means the ratio of the GFP antibody in buffer (1μg of GFP in 500 μl 1XPBS). (C, D). The total protein integrated density values (IDVs) were measured. The abundance of the mutant (GAT-1(G234S) transporter was normalized to the wildtype condition. In C, the total protein abundance was measured by adding up all the three bands run between 90–110 KDa. In both C and D, the total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (***p < 0.001 vs. wt, n=4 different transfections).

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Transfection, SDS Page, Western Blot, Mutagenesis, Cell Culture, Expressing

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: (A) The flow cytometry histograms depict surface expression levels of GAT-1 from HEK293T cells were transfected with wildtype GAT-1YFP (wt), or the mutant GAT-1(G234S)YFP cDNAs or control (insert). Cell surface wild type and mutant GAT-1 stained with polyclonal anti-GAT-1 antibody that was fluorescently conjugated with Alexa Fluor-555. (B) Surface protein was isolated by biotinylating live cells expressing wildtype GAT-1YFP (wt), or the mutant GAT-1(G234S)YFP and analyzed by SDS-PAGE. The membranes were visualized by polyclonal anti-GAT-1 protein. (C) The normalized relative fluorescence intensity (FI) (C) or the surface protein intensity values (IDVs) (D) were normalized to those obtained with wildtype GAT-1 while the FI or protein IDVs in the wildtype was arbitrarily taken as 1 in each experiment (***p < 0.001 vs. wt, n=4 different transfections). (E). The GABA uptake assay was carried out with 3H radioactive GABA transport in HeLa cells transiently expressing the wildtype or the mutant GAT-1YFP for 48 hrs. GABA flux was measured after 15 min transport at room temperature (**p < 0.01 vs. wt, n=6 for wildtype and 7 for the mutant which represents number of transfections).

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Flow Cytometry, Expressing, Transfection, Mutagenesis, Staining, Isolation, SDS Page, Fluorescence

Journal: Stem Cells International

Article Title: Purinergic Signaling Pathway in Human Olfactory Neuronal Precursor Cells

doi: 10.1155/2019/2728786

Figure Lengend Snippet: Detection of P2 receptors in the clonal culture of the hOE. Cloned cells were scraped with a RIPA buffer, and cell lysates were assayed by Western blot to immunodetect P2 receptor subtypes. Representative chemiluminescent bands corresponding to P2X receptors are shown in (a) and to P2Y receptors in (b). (c) shows antibody specificity controls assessed by omission of primary antibody (P2X3) or by preadsorption of anti-P2X5 and anti-P2Y2 with the corresponding blocking peptides. The molecular weights of sample bands were corroborated using biotinylated molecular weight standards. (d) shows immunodetection of P2Y2 and P2Y4 receptors in both the fresh exfoliate (FE) sample and the clonal culture (CC) from the hOE.

Article Snippet: The primary antibodies were as follows: P2X1 (PVDF; Abcam ab81122; 1 : 1500), P2X2 (PVDF; Abcam ab10266; 1 : 500), P2X3 (PVDF; Abcam ab90905; 1 : 1000), P2X4 (PVDF; Alomone APR-002; 1 : 1000), P2X5 (NITRO; Alomone APR-027; 1 : 500), P2X7 (NITRO; Alomone APR-004; 1 : 500), P2Y2 (PVDF; Alomone APR-010; 1 : 1000), P2Y4 (PVDF; Alomone APR-006; 1 : 1000), P2Y6 (NITRO; Alomone APR-011; 1 : 500), and P2Y11 (NITRO; Santa Cruz sc-98600; 1 : 500).

Techniques: Clone Assay, Western Blot, Blocking Assay, Molecular Weight, Immunodetection