Figure 5. Peroxiredoxin 2 is consistently downregulated in mutant mice and a cell culture model of PSACH. A: Protein extracted from the xiphoid cartilage of 3-week-old wild-type (wt) and mutant (mut/mut) mice were analyzed by SDS-PAGE and Western blotting using an antibody against PRDX2 (∼22 kDa). B: Densitometry analysis on the Western blots conﬁrmed that PRDX2 expression is reduced in the cartilage of mutant mice (mut/mut) compared to wild-type animals (wt). C: Cell lysates from HT1080 cells transfected with GFP alone (GFP), GFP-COMP wild-type (WT), and GFP-COMP mutant (MUT) were obtained either 48 or 72 hr after conﬂuence and analyzed by SDS-PAGE and Western blotting using an antibody against PRDX2 (∼22 kDa) and normalized to β actin (∼42 kDa). D: Densitometry analysis on the Western blots conﬁrmed that PRDX2 expression is reduced in cells transfected with the mutant protein compared to those transfected with the wild-type protein. E: Reduced PRDX2 expression in mutant mice cartilage was also conﬁrmed by IHC on the tibial growth plates of 3-week-old mice. PRDX2 appears to be expressed by hypertrophic chondrocytes in the growth plate of wild-type mice, while its expression is much fainter in the same zone of the mutant mice growth plate. wt, wild-type mice; mut/mut, homozygous for D469del; VIF, vascular invasion front; Hz, hypertrophic zone; Pz, proliferative zone; ∗P < 0.05; ∗∗P < 0.005; ∗∗∗P < 0.001. hypothesized that the expression of Comp D469del would cause ER stress and a UPR. However, qRT-PCR and Western blot anal- ysis both failed to reveal any increases in the relative expression of key chaperone proteins at 5 days and 3 weeks of age (Table 1), which was conﬁrmed by extensive microarray studies performed at newborn and 5 days of age (Supp. Table S1 and S2). This is the ﬁrst study to unequivocally establish the relative expression lev- els of chaperone proteins in PSACH chondrocytes and to establish that there is no conventional UPR due to the expression of mutant COMP D469del. Previous studies using patient cartilage or cul- tured patient chondrocytes have shown that mutant COMP is asso- ciated with various chaperone proteins, such as CRT, PDI, Grp94, Erp72, and Grp78/BiP, in distended cisternae of the rER, both in vivo and in vitro [Hecht et al., 2001; Vranka et al., 2001]. Furthermore, 228 HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012