Journal: Neurobiology of disease

Article Title: Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

doi: 10.1016/j.nbd.2016.01.002

Figure Lengend Snippet: Fig. 1. Striatal GDNF expression. (A) GDNF protein levels (pg/mg protein) measured by ELISA in rat striata 5 weeks after PBS (sham) or viral vector (AAV-tetON-GFP or AAV- tetON-GDNF) injection. Viral vector injected rats were treated with different doses of DOX (0.01, 0.03, 0.5 or 3 mg/ml) in the drinking water. The striatal GDNF levels in AAV- tetON-GDNF injected rats receiving 0.5 mg/ml DOX were 3 times higher than in sham rats and AAV-tetON-GFP-injected rats receiving 0.01 and 3 mg/ml DOX. In AAV-tetON- GDNF injected rats receiving 3 mg/ml DOX, GDNF levels were 12 times higher than in sham and AAV-tetON-GFP-injected rats. (B–I) Immunohistochemistry for GDNF in the striatum of sham rats (B) and viral vector injected rats treated with 0.5 (F) and 3 mg/ml DOX (D, H). (C, E, G and I) High-power magnification microphotographs of B, D, F and H, respectively. GDNF staining was not detected in the striatum of sham rats (C) and AAV-tetON-GFP-injected rats treated with 3 mg/ml DOX (E). Sparse GDNF positive cells were detected in the striatum of AAV-tetON-GDNF-injected rats treated with 0.5 mg/ml DOX (G). Striatal tissue was intensely immunoreactive for GDNF in AAV-tetON-GDNF- injected rats treated with 3 mg/ml DOX (H, I). Scale bar in H (for B, D, F and H), 800 μm; in I (for C, E, G and H), 50 μm.

Article Snippet: Floating sections were immersed for 30 min in 3% H2O2 to inactivate endogenous peroxidase, and incubated for 60min at RT in 4% normal donkey serum (NDS, Jackson ImmunoResearch,West Grove, PA) in PBS, containing 0.05% Triton X-100 (TX-100, Sigma), and overnight in PBS containing 2% NDS and a goat anti-GDNF polyclonal antibody (1:500; R&D Systems, Minneapolis, MN).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Injection, Immunohistochemistry, Staining

Journal: Neurobiology of disease

Article Title: Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

doi: 10.1016/j.nbd.2016.01.002

Figure Lengend Snippet: Fig. 2. (A) Western-blot for native TH (TH) and TH phosphorylated at serine 40 (THp40) in whole striatal extracts. (B) Western-blot for DAT in whole striatal extracts and plasma membranes of striatal synaptosomes. (C) Striatal DA uptake. Both TH and THp40 were decreased in the striatum of AAV-tetON-GDNF injected rats treated with 3 mg/ml DOX. No differences were found in the total and membrane DAT levels between sham rats and viral vector injected rats treated with different DOX doses. However, the striatal DA uptake was significantly reduced in AAV-tetON-GDNF injected rats receiving 0.5 and 3 mg/ml DOX, returning to normal levels after 20 days without DOX. β-act, β-actin; synt, syntaxin.

Article Snippet: Floating sections were immersed for 30 min in 3% H2O2 to inactivate endogenous peroxidase, and incubated for 60min at RT in 4% normal donkey serum (NDS, Jackson ImmunoResearch,West Grove, PA) in PBS, containing 0.05% Triton X-100 (TX-100, Sigma), and overnight in PBS containing 2% NDS and a goat anti-GDNF polyclonal antibody (1:500; R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Clinical Proteomics, Injection, Membrane, Plasmid Preparation

Journal: Neurobiology of disease

Article Title: Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

doi: 10.1016/j.nbd.2016.01.002

Figure Lengend Snippet: Fig. 3. Effects of GDNF over-expression on DAT–DAT interaction. (A) Western-blot under non-reducing conditions revealed a weak band at 150 kDa, besides that at 75 kDa, in the striatum of sham rats (lane 1), AAV-tetON-GFP-injected rats treated with 3 mg/ml DOX (lane 2), and AAV-tetON-GDNF-injected rats treated with 0.03 mg/ml DOX (lane 3). The high weight band was significantly more intense in labeling and that at 75 kDa became weaker in AAV-tetON-GDNF-injected rats receiving 0.5 and 3 mg/ml DOX (lanes 4 and 5) compared with sham rats (p b 0.01). (B) DAT–DAT in situ proximity ligation assay (PLA, left) combined with TH immunofluorescence (center) in the striatum of sham rats and viral vector-injected rats treated with 0.5 mg/ml DOX. The quantitative analysis (on the right) showed an increase in both number (top) and size (bottom) of PLA dots in AAV-tetON-GDNF-injected rats treated with 0.5 mg/ml DOX compared with sham rats and AAV-tetON-GFP-injected rats treated with 0.5 mg/ml DOX (C and D; p b 0.01). Arrows indicate that DAT–DAT PLA signals (red) localize in DA-terminals (green). Quantitative analysis was performed in 5 animals per group (see the Material and methods section). In the size diagram, the numbers indicate the average size (pixels) of PLA signals. Each dot corresponds to 8 PLA signals in a field of 200 μm2 from a representative animal. β-act, β-actin. Scale bar in B, 5 μm.

Article Snippet: Floating sections were immersed for 30 min in 3% H2O2 to inactivate endogenous peroxidase, and incubated for 60min at RT in 4% normal donkey serum (NDS, Jackson ImmunoResearch,West Grove, PA) in PBS, containing 0.05% Triton X-100 (TX-100, Sigma), and overnight in PBS containing 2% NDS and a goat anti-GDNF polyclonal antibody (1:500; R&D Systems, Minneapolis, MN).

Techniques: Over Expression, Western Blot, Injection, Labeling, In Situ, Proximity Ligation Assay, Plasmid Preparation

Journal: Neurobiology of disease

Article Title: Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

doi: 10.1016/j.nbd.2016.01.002

Figure Lengend Snippet: Fig. 4. GDNF over-expression modifies DAT–α synuclein but not DAT–D2R interaction. (A) Immunoprecipitation for DAT (IP-DAT) and immunoblotting for α-synuclein (IB-α Syn) and DAT (IB-DAT). The quantity of co-immunoprecipitated α-synuclein was higher in AAV-tetON-GDNF-injected DOX (0.5 and 3 mg/ml)-treated rats (lanes 4 and 5) than in sham rats and AAV- tetON-GFP-injected DOX (3 mg/ml)-treated rats (lanes 2 and 3; p b 0.01). Lane 1 (IgG), control immunoprecipitation using non-immune IgG. (B) PLA for DAT and α-synuclein in the striatum of AAV-tetON-GFP-injected rats (left) and AAV-tetON-GDNF-injected rats (right) treated with 0.5 mg/ml DOX. The PLA analysis showed that GDNF over-expression increases the number and size of PLA dots (p b 0.01 vs. AAV-tetON-GFP). (C) Immunoprecipitation for D2R (IP-D2R) and immunoblotting for DAT (IB-DAT) and D2R (IB-D2R). (D) PLA for DAT and D2R in the striatum. No differences were found in the levels of co-immunoprecipitated DAT among sham rats and AAV-tetON-GFP- and AAV-tetON-GDNF-injected DOX-treated rats, and in the PLA analysis between both viral vector-injected DOX-treated rats. In (B and D), n = 5 animals/group, the numbers in the size diagram indicate the average size (pixels) of PLA signals. Each dot corresponds to 8 PLA signals in a field of 200 μm2 from a representative animal. Scale bar in B and D, 5 μm.

Article Snippet: Floating sections were immersed for 30 min in 3% H2O2 to inactivate endogenous peroxidase, and incubated for 60min at RT in 4% normal donkey serum (NDS, Jackson ImmunoResearch,West Grove, PA) in PBS, containing 0.05% Triton X-100 (TX-100, Sigma), and overnight in PBS containing 2% NDS and a goat anti-GDNF polyclonal antibody (1:500; R&D Systems, Minneapolis, MN).

Techniques: Over Expression, Immunoprecipitation, Western Blot, Injection, Control, Plasmid Preparation

Journal: Cell reports

Article Title: Species-specific MARCO-alphavirus interactions dictate chikungunya virus viremia

doi: 10.1016/j.celrep.2023.112418

Figure Lengend Snippet:

Article Snippet: Surface expression of MARCO and mMxra8 was assessed by flow cytometry with monoclonal antibody ED31 (4 μg/mL; Bio-Rad MCA1849; RRID: AB_2140591), polyclonal human MARCO antibody (1 μg/mL; R&D AF7586), or monoclonal murine Mxra8 antibody (4 μg /mL; a gift from M. Diamond, Washington University), followed by secondary antibodies: phycoerythrin (PE)-anti-rat IgG1 antibody (BioLegend 407407; RRID: AB_2565987), fluorescein isothiocyanate (FITC)-anti-goat IgG antibody (Jackson ImmunoResearch 705–096-147; RRID: AB_2340402), and FITC anti-Armenian hamster IgG antibody (BioLegend 405502; RRID: AB_315020), respectively.

Techniques: Control, Virus, Recombinant, Mutagenesis, Transfection, Gene Expression, Clone Assay, Plasmid Preparation, Software, Flow Cytometry