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. 2017 Jun 7;7:2918. doi: 10.1038/s41598-017-03112-1

Figure 5.

Figure 5

RIPK1 induces ROS accumulation and cell death in the absence of RIPK3 and caspase activities. (A and B) HeLa cells were transfected with an expression vector for GFP-tagged RIPK1 or GFP together with 20 µM zVAD, incubated for 24 h, and stained with CellROX dye or Annexin V-APC and 7-AAD, then analyzed on a flow cytometer. (C) Proteins expression of TAK1 and RIPK1 were analyzed by immunoblotting. β-actin is shown as a loading control. (D and E) HeLa cells were transfected with siRNA targeted against TAK1 gene, incubated for 24 h and then transfected with an expression vector for GFP-tagged RIPK1 or GFP together with 20 µM zVAD, incubated for another 24 h, and stained with CellROX dye or Annexin V-APC and 7-AAD, then analyzed on a flow cytometer.