Fig. 6.

Loss of MGRN1 results in the accumulation of insoluble, high molecular weight (multiubiquitinated) forms of TSG101. Sequential extraction of brain proteins from 1- and 6-month-old wild-type (wt) and Mgrn1 null mutant mice was performed to isolate proteins based on their solubility (proteins in Triton X-100 fractions are more soluble than those in SDS fractions). Fractions were subjected to IB to detect unmodified and ubiquitinated TSG101 (TSG101-(Ub)_n). The same blots were imaged for 1 min (top panels) and 10 min (bottom panels), and blotted for GAPDH as a loading control. In the brains of young animals, multiubiquitinated TSG101 was predominantly observed in the SDS fraction. In older animals, multiubiquitinated TSG101 was observed in both fractions, although more was present in the SDS fraction in the brains of Mgrn1 null mutants than in wild-type mice.