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Since previous studies demonstrated a role for CHGB in supporting neurite outgrowth of cultured cells (34–36), we examined the effects of CHGB transgenic mouse lines that overexpressed human CHGB m RNA species at similar levels in the spinal cord as assessed by quantitative real time RT-PCR.These transgenic mice also exhibited similar excess CHGB protein levels in the microsomal fraction of the spinal cord when examined at 210 days of age ( showed no deficiency on the rotarod test or on body weight loss until 500 days of age (data not shown).Immunoprecipitates with anti-FLAG affinity gel (IP-FLAG) were immunoblotted using anti-HA or anti-SOD1 antibody.(B) DNA construct of genomic CHGB containing 5.1 kb of promoter and 13.7 kb of coding sequence.Arrowheads indicate CHGB accumulation and SOD1 Additional evidence of defective binding of CHGB variants (L413 and R230) with mutant SOD1 or misf SOD1 was obtained by immunofluorescence confocal microscopy of Neuro2a cells after transfection of vectors encoding mouse Cg B or human CHGB variants with SOD1 species. To further investigate the localization of CHGB WT and variants in the context of overexpression of human SOD1 species, Neuro2a cells were co-transfected with HA-CHGB species and FLAG-SOD1 species, and then analyzed by double immunofluorescence using antibodies against trans-Golgi marker (TGN38) or synaptic vesicle marker (synaptophysin).
Neuro2A cells were co-transfected with FLAG-tagged SOD1 and HA-tagged CHGB.
A few years ago we reported that a common chromogranin B (CHGB) variation was associated with higher risk to develop ALS and with earlier age of onset in both sporadic and familial ALS cases in cohorts of French-Canadian origin (28).
However, these results have not been corroborated by other groups that reported a lack of association of , detected only in few ALS cases but not in control individuals (28).
We carried out transient co-expression assays in Neuro2a cells using plasmid vectors coding for mouse chromogranin B (m Cg B) or various human CHGB species tagged with hemagglutinin (HA) at the carboxy terminus together with vectors coding for wild type (WT) or SOD1.
The genomic CHGB fragments included 5.1 kb of CHGB promoter region and 13.7 kb of exon/intron sequences (Fig. Total cell lysates from transfected cells were fractionated by two-dimensional gel electrophoresis and then transferred to a membrane for immunodetection of CHGB. Neuro2A cells were co-transfected with FLAG-tagged SOD1 and HA-tagged CHGB.