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. 2021 Jan 27;108(2):295–308. doi: 10.1016/j.ajhg.2021.01.006

Figure 3.

Figure 3

Efficient knockdown of p.Pro347Ser RHO expression in vitro

(A) Immunoblot for rhodopsin protein expressed in Pro347Ser and WT RHO HeLa clones transfected with SpCas9_gRNA1 and VQRHF1-SpCas9_gRNA5 plasmids and control plasmids (SpCas9 and VQRHF1-SpCas9). 4D2 antibody was used to detect rhodopsin. The immunoblotting was normalized with anti-beta-actin antibody.

(B) Densitometric quantification of rhodopsin protein level normalized to beta-actin after editing. The experiment was performed in triplicate and is presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.

(C) Pro347Ser and WT RHO HeLa clones were transfected with SpCas9_gRNA1 and VQRHF1-SpCas9_gRNA5 plasmids and control plasmids (SpCas9 and VQRHF1-SpCas9). The relative quantity (RQ) was calculated with the 2-ΔΔCT quantification and is reported in the y axis. Each sample was run in triplicate. ∗∗p < 0.01.

(D) Lactate dehydrogenase (LDH) assay of Pro347Ser RHO HeLa clone transfected with SpCas9_gRNA1 and VQRHF1-SpCas9_gRNA5 plasmids and control plasmids (SpCas9 and VQRHF1-SpCas9). Mock-transfected Pro347Ser RHO HeLa cells and WT RHO HeLa cells were used as positive and negative controls. The experiment was performed in triplicate and is presented as mean ± SEM. ∗p value < 0.05.