Figure 7

Inhibition of UCH-L1 protects podocytes from TNF-induced necroptosis. Podocytes stably transfected with an shRNA construct that causes permanent knockdown of UCH-L1 (shUCH-L1) or with a scrambled negative control shRNA (shCtr) were treated with 100 ng/ml TNF in the presence of 50 μM zVAD-fmk or vehicle for 3 h before loss of membrane integrity as a marker for cell death was measured by trypan blue staining. Asterisks indicate statistical significance (t-test), *p < 0.05. The Western blot below was performed to demonstrate the permanent knockdown of UCH-L1 in shUCH-L1 podocytes, but not in shCtr podocytes. UCH-L1 was detected with mAb UCH-L1, detection of actin served as a loading control. For each stable transfectant, lysates from four independent flasks were analyzed.