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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.The disease onset, as defined by 30% loss of maximum recorded time on rotarod (37,38), occurred at mean age of 365.7 ± 3.0 days in SOD1 mice (Fig. Furthermore, we have stratified the tested group of mice according to their sex status and determined whether sex had an effect of disease onset by analyzing rotarod performance. To investigate the degeneration of spinal α-motor neurons, lumbar cord sections at 345 days of age were immunostained with both anti-Ch AT and anti-Neu N antibody.The number of α-motor neurons, which were positive for both Ch AT and Neu N, in SOD13–4) was analyzed.Some of the HA-CHGB WT was co-localized with the accumulation of synaptic vesicle marker in cells co-transfected with SOD1.Arrowheads indicate CHGB accumulation merged with TGN38.

The percentages of CHGB vesicles merged with TGN-38 marker were analyzed. Arrowheads indicate CHGB accumulation merged with synaptophysin. Arrowheads indicate CHGB accumulation merged with TGN38.(B) The reduced sizes of α-motor neurons (α-MNs) in SOD13–4) was analyzed.(B) The reduced sizes of α-motor neurons (α-MNs) in SOD1 mice (Fig. This effect was more pronounced in female than male mice (Fig. The neuromuscular junctions of gastrocnemius muscles were examined at 345 days of age.Double arrowheads indicate CHGB accumulation not merged with synaptophysin. (C) Impaired promotion of CHGB had no effect of the mutant SOD1 secretion.However, the expression of CHGB (WT, L413 and R230) and mouse Cg B did not promote the secretion of endogenous mouse SOD1 in cells (data not shown).

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Arrowheads indicate CHGB accumulation and SOD1 in cultured cells.