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. 2013 Nov 7;23(7):1709–1722. doi: 10.1093/hmg/ddt560

Figure 5.

Figure 5.

Biosynthetic effects of Col4a1 and Col4a2 mutations. (A) Representative photomicrographs of Col4a1+/+ and Col4a1+/mut mouse embryonic fibrobalsts (MEFs) co-immunolabeled for COL4A1 and HSP47, a collagen-specific chaperone residing in the ER. Col4a1 mutations lead to variable increase in co-localization of COL4A1 and HSP47, suggesting that some mutations lead to more severe intracellular accumulation than others. (B–G) Western blot and densitometric analyses of intracellular and extracellular COL4A1 and COL4A2 levels in MEFs. (B) Soluble intracellular COL4A1 levels (or COL4A2 for Col4a2+/G646D) were normalized to an actin loading control and compared with samples from Col4a1+/+ littermates loaded on the same gel under reducing conditions. (C) Extracellular COL4A1 levels were determined by analyses of conditioned media from the same cell cultures used in (B). Loading volumes were normalized to the total protein concentration of the cellular fraction and the conditioned media from Col4a1+/mut cells were compared with that of cells from Col4a1+/+ littermates that were loaded on the same gel. In a separate experiment, laminin labeling was used to demonstrate that normalization to total protein concentration returned consistent results (Supplementary Material, Fig. S3). For (B) and (C), we used three to five independent MEF lines isolated from individual embryos for each mutant line. Values were compared using Student's t-test (*P < 0.05; **P < 0.01) and are presented as mean ± SEM. (D–G) To compare strains directly, reduced total intracellular or conditioned media from all strains were loaded on a single gel and probed for COL4A1 or COL4A2. We used five independent MEF lines derived from different embryos for each mutation and the values amongst strains run on the same gel were compared using a one-way ANOVA followed by a Tukey's post hoc test (†P < 0.01), arrow indicates a COL4A1 band of higher molecular weight. (H) Representative photomicrographs of western blot analyses of detergent-soluble and-insoluble intracellular COL4A1 in Col4a1+/+ (wt) and mutant (mut) MEFs showing variability in the proportions of intracellular COL4A1 monomers (M) and COL4A1-containing heterotrimers (H) in MEFs with different Col4a1 mutations. We used three to five independent MEF lines derived from different embryos per mutation.