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. 2021 Sep 21;16(9):e0257537. doi: 10.1371/journal.pone.0257537

Fig 3. Targeting B2AR as a model GPCR.

Fig 3

A. Western Blot for Cas9 expression levels in three cell lines: Wild-type HEK293T, HEK293T + dCas9-AID*Δ as a pool, and the single cell clone of HEK293T + dCas9-AID* selected for the highest expression of Cas9. Housekeeping protein vinculin is used as reference. Blots were cropped where indicated by the arrow. Full uncropped blots are provided in S1 Fig. B. Clone HEK293T dCas9-AID*Δ was infected with 6xCRE-mCherry reporter system and single cell clones were evaluated. The most homogeneous clone for mCherry expression was selected. mCherry red fluorescence was measured with high content imaging using a cell incubator imaging system. Cells treated with and without isoproterenol were monitored for 30 hours after stimulation. C. mCherry fluorescence intensity of the same single cell clone as panel B, 24 hours after isoproterenol treatment or in its absence. D. B2AR and B1AR mRNA expression 72h after transfection of 10pmol of siRNA. The mean of 4 replicates is shown. E, Left, mCherry fluorescence intensity of cells transfected with control siRNA or siRNA against B2AR. Right, mCherry fluorescence intensity of cells transfected with control siRNA or siRNA against B1AR. In both cases, cells were stimulated with isoproterenol after siRNA treatment. F. mCherry fluorescence intensity of knock-out cells for B2AR, with and without stimulation of isoproterenol.