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Fig. 6. SSPN induces aggregation of the SGs. (A) Schematic diagram to illustrate the experimental procedure. Following two 1% digitonin solubilizations (Supe #1 and Supe #2), insoluble protein aggregates were extracted using 3% SDS homogenization buffer. (B) Non-phenotypic and phenotypic SSPN-Tg (SSPN-Tg) muscle solubilized fractions contain more DGC proteins than non-Tg (Non-Tg) controls. Digitonin solubilized proteins (60 µg) were analyzed by 12% SDS-PAGE, transferred to nitrocellulose, and analyzed for components of the DGC by immunoblot analysis. Coomassie Blue (CB) stain was used to confirm equal loading of protein samples. (C) SDS solubilized proteins (60 µg) from non-Tg (Non-Tg) and SSPN-Tg (SSPN-Tg) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. Representative data are shown from non-phenotypic (line 31.6) and phenotypic (line 31.7) SSPN-Tg mice. Nitrocellulose membranes were separately stained for 
-DG and 
-DG, -SG, -SG and 
-SG, as well as the SSPN transgene (hSSPN). Equal protein loading was confirmed by Coomassie Blue (CB) stain. (D) Quantification of transgene (hSSPN) solubilization. Percentage of solubilized protein was determined by relative densitometry. Relative levels of hSSPN in the soluble and insoluble fractions were analyzed for the phenotypic and non-phenotypic samples. Data is presented as percentages relative to the total level of transgene expression in the soluble and insoluble fractions combined. (E) SSPN homo-oligomerization in non-phenotypic and phenotypic insoluble fractions. SDS solubilized proteins (60 µg) from non-phenotypic and phenotypic SSPN-Tg muscle were analyzed by SDS-PAGE under reducing (R) or non-reducing (NR) conditions and transferred to nitrocellulose. Membranes were probed with antibodies recognizing transgene (hSSPN) expression. Higher-order SSPN oligomers were present only in extracts from phenotypic SSPN-Tg muscle.
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