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. 2021 Sep 27;11(20):9752–9771. doi: 10.7150/thno.63806

Figure 2.

Figure 2

DUB knockout library kit-based screening for USPs altering SLC35F2 protein stability through Western blot analysis. (A) HeLa cells transfected with sgRNAs individually targeting entire set of genes encoding USPs along with Cas9 were subjected to western blot analysis to determine endogenous SLC35F2 protein levels. HeLa cells co-transfected with Cas9 and scrambled sgRNA served as mock controls. GAPDH was used as a loading control. (B) Relative protein expression of SLC35F2 was quantified using ImageJ software and represented as a bar graph. (C) A Venn diagram showing the overlapping candidates from our dual-screening approach (cell viability assay and western blot analysis). (D) Overlapping candidates were validated by western blot analysis. The sgRNAs targeting USP19, USP32, and USP49 were transfected in HeLa cells and a western blot was performed with the indicated antibodies. (E) An immunofluorescence staining assay was performed to analyze the co-localization behaviors of USP19, USP32, and USP49 with respect to SLC35F2 in HeLa cells. Because specific antibodies against USP19, USP49, and SLC35F2 are derived from the same species, we transfected HeLa cells with either Flag-USP19 or Flag-USP49 and then performed staining with an anti-Flag and SLC35F2-specific antibodies. Scale bar = 25 µm.