Journal: Molecular Therapy Oncolytics

Article Title: Adoptive T cell immunotherapy for medullary thyroid carcinoma targeting GDNF family receptor alpha 4

doi: 10.1016/j.omto.2021.01.012

Figure Lengend Snippet: P4-10 CAR T cells specifically respond to GFRα4 protein in vitro (A) Schematic of CAR construct inserted downstream of an EF1a-derived promoter within a 3 rd -generation lentiviral vector plasmid. (B) Primary human T cells were activated using anti-CD3/anti-CD28-coated beads followed by transduction with lentiviral vectors encoding the indicated CAR genes or were left non-transduced (NTD). Seven days later, T cells were stained with biotinylated F(ab′) 2 -specifc goat anti-mouse or donkey anti-rabbit followed by streptavidin-AF647 and analysis by flow cytometry. (C) NFAT-GFP reporter Jurkat cells expressing no CAR (NTD), 19bbz, or the P4-10bbz CAR were cultured in wells that had been coated overnight with GFRα1, GFRα2, GFRα3, GFRα4, and OKT3 proteins. Following overnight culture, cells were analyzed by flow cytometry for GFP expression. A representative of 3 independent experiments is shown. (D and E) NFAT-GFP reporter Jurkat cells (D) or primary human T cells (E) expressing no CAR (NTD), or the P4-10bbz CAR were cultured in wells that had been coated overnight with OKT3 or GFRα4 (bound) or in media with soluble GFRα4 (soluble). Following overnight culture, cells were analyzed by flow cytometry for GFP expression (*p < 0.001 compared to media control, ANOVA, Dunnett’s multiple comparisons test). Representatives of 2 independent experiments each are shown. Error bars indicate 1 standard deviation of the mean.

Article Snippet: Expression of GFRα1, GFRα2, and GFRα3 was assessed using polyclonal rabbit anti-GFRα1, anti-GFRα2, and anti-GFRα3 (Sino Biologicals, Wayne, PA, USA) followed by AF647-conjugated goat anti-rabbit antibody (Thermo Fisher Scientific).

Techniques: In Vitro, Construct, Derivative Assay, Plasmid Preparation, Transduction, Staining, Flow Cytometry, Expressing, Cell Culture, Standard Deviation

Journal: The Journal of Neuroscience

Article Title: Death Receptors and Caspases But Not Mitochondria Are Activated in the GDNF- or BDNF-Deprived Dopaminergic Neurons

doi: 10.1523/JNEUROSCI.1877-08.2008

Figure Lengend Snippet: FAIML associates with Fas and inhibits its apoptotic activity. A, Immunoblot of the midbrain cultures maintained with GDNF or BDNF for 5 DIV. Lysates of mouse 3T3 cells or mouse cortex were included as negative or positive controls, respectively. Reprobing of the filter with antibodies to actin shows the total protein loading. B, Coimmunoprecipitation of FAIML with Fas. GDNF- or BDNF-maintained midbrain cultures (5 DIV) were transfected with expression plasmid for Fas for 2 d and immunoprecipitated with biotin-anti-Fas antibody or control antibody (biotin-GFRα2). The filter was probed for coprecipitated endogenous FAIML (∼23 kDa) and reprobed for Fas (about 45 kDa). IP, Immunoprecipitation. C, FAIML and Fas interact functionally in the DA neurons. GDNF- or BDNF-maintained neurons were transfected with expression plasmids for FAIML, Fas, FAIML and Fas together (FAIML/Fas), or empty pcDNA3 plasmid (vector) (all together with the plasmid for EGFP) and maintained with (GDNF+, BDNF+) or without (GDNF−, BDNF−) neurotrophic factors for 3 d. The numbers of fluorescent neurons counted at the end of the experiment were normalized to those counted at the factor deprivation. Transfected non-TH neurons, identified by post hoc TH immunostaining, were subtracted from the counts. The mean ± SEM of four independent experiments is shown. Statistical significance of the differences was estimated by one-way ANOVA and post hoc Tukey's honestly significant difference test. The null hypothesis was rejected at p < 0.05.

Article Snippet: Fas was precipitated by biotin-coupled anti-Fas antibody (BD Biosciences) or control biotin-coupled anti-GFRα2 antibody (BD Biosciences), and the immunocomplexes were collected with NeutrAvidin (Thermo Fisher Scientific).

Techniques: Activity Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Immunostaining