Protein Expression Regulation by TetR-DOZI System

Western blotting for SpCas9 and T7 RNAP was described previously ^{13 (link)}. To determine regulation of target protein expression by the TetR- or TetR-DOZI-aptamer system, parasites cultured with 0 nM or 50 nM aTc for 72 h were saponin-lysed, washed with 1 × PBS, and proteins solubilized in lysis buffer containing 4% sodium dodecyl sulfate (SDS) and 0.5% Triton X-114 in 1 × PBS. Protein extracts were mixed with loading buffer containing SDS and dithiothreitol (DTT) and loaded onto Mini-PROTEAN TGX Precast Gels (4–15% gradient; Bio-Rad 4,561,084) in Tris–Glycine buffer. After separation by polyacrylamide gel electrophoresis (PAGE), proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell system (Bio-Rad) per the manufacturer’s instructions and blocked with 100 mg/mL skim milk in 1 × TBS/Tween 20. PVDF membrane-bound proteins were first probed with mouse anti-HA (1:3000; Sigma, H3663) and rabbit anti-GAPDH (1:5000; Abcam, AB9485) primary antibodies and anti-mouse (1:5000; Thermo Fisher Scientific, 62–6520) and anti-rabbit (1:5000; Cell Signaling, 7074S) horseradish peroxidase (HRP)-conjugated secondary antibodies. Following incubation in SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, PI34080), protein blots were imaged and analysed using the ChemiDoc MP System and Image Lab 5.2.0 (Bio-Rad).