Clone c 20

Nearly confluent cells from the above-described experiments were washed with PBS and harvested in lysis buffer (10 mM Tris–HCl; 1 mM EDTA; 1 mM EGTA; 100 mM NaCl; 1 % Triton X-100; 0.5 % Nonidet P-40, pH 7.4) containing a cocktail of kinase, protease and phosphatase inhibitors (Sigma-Alrich, St. Louis, MO). Cells were scraped, collected, sonicated and then resuspended in 2X SDS loading buffer (100 mM Tris-Cl, pH 6.8 + 4 % SDS, 0.2 % bromophenol blue, 20 % glycerol, 200 mM DTT) before denaturing at 95 °C for 5 min. Total cellular proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore,Darmstadt, Germany). Immunoblotting was performed using antibodies to detect human cFLIP (also known as CFLAR) (rabbit anti-human CFLAR; Sigma Aldrich, St. Louis, MO) and human FAS (clone C-20; Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were stripped and reprobed for cleaved human poly ADP ribose polymerase (PARP) (# 9542, Cell Signaling Technology, Danvers, MA), to verify the apoptotic effect, and β-actin (clone AC-15; Sigma Aldrich, St. Louis, MO) to ensure equivalent protein loading.

IF analysis was as described earlier. Zebrafish cilia were labelled according to the protocol of Jaffe et al. (66 (link)) using a mouse-anti-acetylated tubulin antibody (1:500, clone 6-11B-1, Sigma-Aldrich) and an Alexa 488-labelled secondary antibody. KV outlines were visualized through immunostaining with an antibody against atypical PKC (1:500, clone C-20, Santa Cruz Biotechnologies).