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. 2021 Jan 21;24(2):102076. doi: 10.1016/j.isci.2021.102076

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© 2021 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Generation of genetically engineered CHO cells expressing LOX-1 and/or AT1 or biased AT1 mutants

(A) Overview of two biased AT1 mutants.

AT1mβ, AT1 mutant with β-arrestin-biased signaling; AT1mg, AT1 mutant with G-protein-biased signaling.

(B) Immunofluorescence of CHO cells stably expressing V5-tagged LOX-1 and/or FLAG-tagged AT1 or biased AT1 mutants using anti-V5 or anti-FLAG antibodies. Scale bar (μm).

(C) Quantification of membrane-expressing V5-tagged LOX-1 and FLAG-tagged AT1 or biased AT1 mutants by cell-based ELISA. Absorbance in CHO cells expressing V5-tagged LOX-1 and FLAG-tagged AT1 (CHO-LOX-1-AT1) was normalized to 100% (n = 5, each). Data are represented as mean ± SEM. The differences were determined by one-way analysis of variance (ANOVA) with Bonferroni correction. ^∗p < 0.01 vs. the other types of cells.

(D) In situ proximity ligation assay (PLA) of CHO cells stably expressing V5-tagged LOX-1 and/or FLAG-tagged AT1 or biased AT1 mutants using anti-V5 and anti-FLAG antibodies. Scale bar (μm).

The graph indicates the fluorescence/number of nuclei (% of that in CHO-AT1) (n = 3, each) Data are represented as mean ± SEM. The differences were determined by one-way ANOVA with Bonferroni correction. ^∗p < 0.01 vs.CHO-AT1, CHO-LOX-1, †p < 0.05 vs.CHO-AT1, p < 0.01 vs. CHO-LOX-1.