Manual - New England Biolabs

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8 Luciferase Activity (RLU) 90,000 45,000 80,000 70,000 60,000 50,000 40,000 30,000 20,000 10,000 0 0 800 pM 4 nM 20 nM 100 nM 500 nM RSL1 Concentration 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000 Figure 3: Luciferase expression in response to increasing RSL1 concentrations. NIH 3T3 cells co-transfected with pNEBR-R1 and pNEBR-X1 expressing firefly luciferase were induced with the indicated RSL1 concentrations. Expression was measured 48 hours postinduction. 0 Fold Induction
62 47.5 32.5 25 16.5 A B C 10 50 100 500 0 nM RSL1 62 47.5 32.5 25 16.5 10 50 100 500 0 10 50 100 500 0 nM RSL1 Figure 4: NIH 3T3 (A) and RheoSwitch HEK293-A7 (B) (NEB #C2003) cells were transfected with pNEBR-X1Hygro encoding a HA-tagged protein using TransPass D2 (NEB #M2554). NIH 3T3 cells were also co-transfected with pNEBR-R1. RSL1 was added to transfected cells 16 hours later, and cells were harvested 24 hours after induction (Note: DMSO is added as a control for 0 nM RSL1). Harvested cells were lysed, and equivalent amounts of protein (as determined by Coomassie Stain) were electrophoresed and transferred to nitrocellulose. An immunoblot assay was performed on the filter using anti-HA as primary antibody and detected using anti-mouse secondary antibody and reagents. A longer exposure of the HEK293-A7 immunoblot is shown (C) to illustrate that no protein is detected in the absence of RSL1. 9
Page 1: Catalog #E3000S Store at -20°C Rhe
Page 4 and 5: 2 Supplied Components: The RheoSwit
Page 6 and 7: 4 Introduction: The RheoSwitch ® M
Page 8 and 9: 6 amounts of RSL1 (Figure 4). Even
Page 12 and 13: Luciferase Activity (RLU) 10 100,00
Page 14 and 15: 12 Gaussia Luciferase (GLuc) Contro
Page 16 and 17: 14 To induce expression, RSL1 ligan
Page 18 and 19: 16 4. Add RSL1 ligand to a final co
Page 20 and 21: 18 B. PshA I 3640 BfuA I - BspM I 3
Page 22 and 23: 20 References: 1. Karzenowski, D.,
Page 24: 22 Licensing Information: RheoSwitc

Manual - New England Biolabs

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