Validation of the 3xFLAG/mCherry and 3xHA/eGFP tagged COL4A1 isoforms in vitro and in vivo. (A) Western blot analyses under reducing conditions of lysates and conditioned media collected from primary mouse embryonic fibroblasts (pMEFs) confirmed the presence of 3xFLAG/mCherry-COL4A1 and 3xHA/eGFP-COL4A1 at the expected molecular weights in cell lysates and conditioned media from Col4a1^+/mCherry and Col4a1^+/eGFP reporter lines, respectively. COL4A2 was also detected in both the cell lysate and conditioned medium at levels similar to those observed in control pMEFs, suggesting the tagged proteins do not disrupt the collagen α1α1α2(IV) biosynthesis. β-Actin (ACTB) and fibronectin 1 (FN1) were used as loading controls for lysates and conditioned media, respectively. n = 2 independent experiments. (B) Schematic of an eye (left) with its main anatomical structures labeled. Schematic of a lens (middle) showing structures imaged in C. An enlarged area of the anterior lens including the anterior lens capsule and the lens epithelium (right) showing structures imaged in D. (C) Representative fluorescence images of lens sections from P7 Col4a1^+/mCherry and Col4a1^+/eGFP mice showing endogenous mCherry (magenta) and eGFP (green) signals, respectively, in the entire lens capsule. Sections were counterstained with DAPI (blue). Scale bar = 200 μm. (D) Representative fluorescence images of the anterior lens capsule and lens epithelium from P7 Col4a1^+/mCherry and Col4a1^+/eGFP mice. Scale bar = 20 μm. n = 3 per genotype from 3 litters. Source data are available for this figure: SourceData F2.