Fig. 2. Development and validation of shRNAs for SOCE components. (a) Candidate sequences for Orai1 shRNA, Orai2 shRNA, Orai3 shRNA, TrpC1 shRNA, and Stim1 shRNA are shown with vector information. Each shRNA was cloned in pENSR vector to express under U6 promoter together with a cytomegalovirus (CMV) promoter driving expression of mCherry reporter gene. (b) Fluorescence images of HEK293-T cells co-transfected TrpC1 shRNA (shRNA-mCherry) with TrpC1 full clone (TrpC1-GFP). Top, Scrambled shRNA-mCherry was co-transfected with TrpC1-GFP. Bottom, Candidate 2 of TrpC1 shRNA was co-transfected with TrpC1-GFP. (c) Quantification of TrpC1 mRNA by RT-PCR. Top, Intensity of RT-PCR bands of TrpC1 shRNA (sh1), TrpC1 shRNA 2 (sh2), TrpC1 shRNA3 (sh3) decreased. Bottom, GAPDH. (d) Knock-down rate of Orai1 shRNA candidates compared to scrambled shRNA, Candidate1: 10.4, Candidate2: 27.2, Candidate3: 54.9. (e) Knock-down rate of Orai2 shRNA candidates compared to scrambled shRNA, Candidate1: 26.9, Candidate2: 16.7, Candidate3: 30.6, Cadidate4: 7.1. (f) Knock-down rate of Orai3 shRNA candidates compared to scrambled shRNA, Candidate1: 72.3, Candidate2: 78.3, Candidate3: 11.5, Candidate4: 54.8. (g) Knock-down rate of TrpC1 shRNA candidates compared to scrambled shRNA, Candidate1: 75.6, Candidate2: 86.7, Candidate3: 27.0. (h) Knock-down rate of Stim1 shRNA candidates compared to scrambled shRNA, Candidate1: 43.2, Candidate2: 22.5, Candidate3: 52.4.
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