Additional details on the design and validation of the Kaleidoscope reporter. (A) Intermediate vector to show cloning sites to concatenate the three fusion proteins. The bolded MluI sites were used to transfer them to the Ai9 targeting vector to generate the ROSA-Kaleidoscope plasmid. The Lamp1 sequence is from rat while the MLS and α-Tubulin sequences are from human. See Table 1 for cloning primer sequences and the order of cloning. (B) MLE15 cells transfected with Cre-recombined ROSA-Kaleidoscope to show comparable fluorescence patterns as in HEK 293T cells. Scale: 10 μm. (C) Expected PCR genotyping results of ROSAKaleidoscope littermates. See Table 2 for genotyping primer sequences. (D) Additional examples of colocalization between BFP-Lamp1 and Lamp2 immunostaining (top row) and MLS-mKate2 and Tomm20 immunostaining (bottom row) in P7 CMV-Cre; ROSAKaleidoscope primary lung cells with a high level of pixel-level Pearson coefficient between cognate pairs. Scale: 10 μm. Each symbol represents one image. Asterisk, P < 0.05 (ordinary ANOVA with Tukey test; data are mean and standard deviation). Tomm20 is in the outer mitochondrial membrane whereas MLS targets mKate2 to the mitochondrial matrix, potentially lowering the theoretically maximal Pearson coefficient. (E) Western blots of lungs expressing all three fusion proteins and HEK 293T cells expressing individual fusion proteins. The second largest band specific to the Kaleidoscope lungs might be due to incomplete glycosylation of LAMP1. Both BFP and mKate2 are tagRFP derivatives and detected by the same antibody. Source data are available for this figure: SourceData FS1.
Figure S1.

Additional details on the design and validation of the Kaleidoscope reporter. (A) Intermediate vector to show cloning sites to concatenate the three fusion proteins. The bolded MluI sites were used to transfer them to the Ai9 targeting vector to generate the ROSA-Kaleidoscope plasmid. The Lamp1 sequence is from rat while the MLS and α-Tubulin sequences are from human. See Table 1 for cloning primer sequences and the order of cloning. (B) MLE15 cells transfected with Cre-recombined ROSA-Kaleidoscope to show comparable fluorescence patterns as in HEK 293T cells. Scale: 10 μm. (C) Expected PCR genotyping results of ROSAKaleidoscope littermates. See Table 2 for genotyping primer sequences. (D) Additional examples of colocalization between BFP-Lamp1 and Lamp2 immunostaining (top row) and MLS-mKate2 and Tomm20 immunostaining (bottom row) in P7 CMV-Cre; ROSAKaleidoscope primary lung cells with a high level of pixel-level Pearson coefficient between cognate pairs. Scale: 10 μm. Each symbol represents one image. Asterisk, P < 0.05 (ordinary ANOVA with Tukey test; data are mean and standard deviation). Tomm20 is in the outer mitochondrial membrane whereas MLS targets mKate2 to the mitochondrial matrix, potentially lowering the theoretically maximal Pearson coefficient. (E) Western blots of lungs expressing all three fusion proteins and HEK 293T cells expressing individual fusion proteins. The second largest band specific to the Kaleidoscope lungs might be due to incomplete glycosylation of LAMP1. Both BFP and mKate2 are tagRFP derivatives and detected by the same antibody. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal