Validation of recombinase-dependent, isoform switchable properties of Col4a1 ^Tag1 . (A) Western blot analyses of lysates and conditioned media collected from Col4a1^+/Tag1 (+/T) pMEFs with or without the ubiquitous inducible Cre transgene (R26^CreERT) in the absence or presence of 4-hydroxytamoxifen (4-OHT). In the absence of 4-OHT, the 3xFLAG/mCherry-COL4A1 isoform was detected both in the lysate and media. Following overnight 4-OHT treatment, the 3xFLAG/mCherry-COL4A1 isoform switched to the 3xHA/eGFP-COL4A1 isoform. n = 2 independent experiments. (B) Representative fluorescence images of the anterior lens capsule from P7 Col4a1^+/Tag1 (+/Tag1) mice with or without the inducible Cre transgene (R26-CreERT) following postnatal tamoxifen administration. The anterior lens capsule in Col4a1^+/Tag1 mice without Cre showed endogenous baseline mCherry (magenta) signal. Postnatal tamoxifen induction in Col4a1^+/Tag1;R26^CreERT mice resulted in localization of 3xFLAG/mCherry-COL4A1 and 3xHA/eGFP-COL4A1 (green) in the outer and inner layers of the lens capsule, respectively. The sections were counterstained with DAPI (blue). n = 3 per genotype from 3 litters. Scale bar = 10 μm. (C) Western blot analyses of lysates and conditioned media collected from Col4a1^+/Tag1 (+/T) pMEFs with or without constitutively active Flp (R26^Flpe). As expected, COL4A1 and COL4A2 were detected in both lysates and media. Flp-mediated recombination resulted in isoform switching from 3xFLAG/mCherry-COL4A1 (asterisks) to the smaller 3xFLAG/mCherry-NLS fusion protein as shown in the lysates (arrows). The 3xFLAG/mCherry-NLS protein is predicted to localize intracellularly and was not detected in the conditioned media by the anti-mCherry antibody. The anti-FLAG antibody recognized a non-specific band at molecular weight slightly above that of the 3xFLAG/mCherry-NLS fusion protein. Moreover, a faint band with the same size as the 3xFLAG/mCherry-NLS protein from the lysate was detected in the media, which may have been released by a small number of dead cells. n = 2 independent experiments. (D) Representative fluorescence images of the anterior lens capsule from P7 Col4a1^+/Tag1 (+/Tag1) mice with or without the ubiquitous Flp (R26-Flpe). Flp-mediated recombination eliminated mCherry signal (magenta) from the lens capsule and resulted in intracellular mCherry labeling. GLUT1 (green) marks the plasma membrane confirming intracellular localization. The sections were counterstained with DAPI. n = 3 per genotype from three litters. Scale bar = 10 μm. Source data are available for this figure: SourceData F3.