RNF5 in ER-Related Disorders Figure 6. Long term muscle specific expression of RNF5 results in the formation of vacuoles containing aggregates. A. Schematics depicting transgenic constructs used to overexpress RNF5 in a muscle specific manner. The tTA transcriptional activator is expressed under the control of the Muscle Creatine Kinase (MCK) promoter and is inactive in the presence of doxycyclin. Once doxycyclin is withdrawn, tTA activates the expression of the RNF5 transgene. B. Muscle specific expression of RNF5 transgene in MCK-tTA RNF5 DTg transgenic animals. RNF5 protein levels in vastus lateralis from rtTA and MCK-tTA RNF5 DTg and RNF5 STg mice were monitored by western blot, 20 wk after doxycyclin withdrawal. C. Weight curve comparison of MCK-tTA RNF5-overexpressing DTg animals and their matching controls after doxycyclin withdrawal. The body mass of individual animals was monitored for 20 wk. Graphs represent the change in body mass relative to the original weight of a single animal (n = 5). D. Cross-sections of fresh frozen specimens of vastus lateralis muscles from RNF5 STg (a, c, e, g) or MCK-tTA RNF5 DTg (b, d, f, h) animals 20 wk after doxycyclin withdrawal. H&E (a, b, e, f) and congo red (c, d) stain have been performed on triceps cross-section. Panels f and g represent a higher magnification to better visualize the presence of vacuole containing aggregates (arrows). E. Expression of GRP94 in muscles of MCK-tTA RNF5 DTg animals compared to their control littermates, 6 wk and 20 wk after doxycyclin withdrawal. F. GRP94 immunostaining of vastus lateralis cross- sections from MCK-tTA RNF5 DTg and STg animals 20 wk after doxycyclin withdrawal. doi:10.1371/journal.pone.0001609.g006 PLoS ONE | www.plosone.org 9 February 2008 | Volume 3 | Issue 2 | e1609