Fig. 5

The Y628H substitution prevents Col8a1 trimerization. A: Constructs encoding wild-type (lane 1) or gul^m208 mutant (lane 2) Col8a1 were transcribed and translated in the presence of radiolabeled methionine, and the products analyzed as described under the Experimental Procedures section. Col8a1 monomers and trimers are indicated. Cotranscription and translation of half the amounts of wild-type and mutant construct together does not prevent trimerization (lane 3). The approximate location of molecular weight markers (kDa) is noted. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) to assess the splice status of xbp1 in wild-type embryos and gul^m208 mutants at 24 hours postfertilization (hpf). Splicing of xbp1 is not observed in gul^m208 mutants but occurs after incubation of wild-type embryos in tunicamycin. U, unspliced; S, spliced. Amplification of the gene spt was performed in parallel to confirm that equal amounts of cDNA were used for each condition.