ZFP36 promotes RIP1 assembly in a high molecular weight complex containing caspase8 and FADD. A, B) Following 24 hours of empty vector/ZFP36 transfection, HEK293T cells were further treated for 24 hours in the presence of 20 μM Z-VAD-FMK, in order to block caspases’ activity and facilitate the recovery of the Ripoptosome, as described in Tenev et al. . A positive control for Ripoptosome assembly was added in the co-immunoprecipitation experiments depicted in figure B, where HEK293T were treated for 24 hours with 100 μM Etoposide and 20 μM Z-VAD-FMK. A) 2 milligrams of lysates from either empty vector (E.V.) or ZFP36 transfected cells (ZFP36) were loaded on a Sepharose column and fractions were eluted at defined timing and then loaded on polyacrylamide gel to perform Western blotting. In the presence of ZFP36, RIP1 was detected in higher molecular weight fractions (54–71), while this was less evident in E.V. samples. A positive control for RIP1 was loaded for each sample series (C+). Molecular weight markers were eluted through the Sepharose column in order to identify the 2MDa fractions. B) Ripoptosome recovery was carried out by immunoprecipitating caspase8, while immunoprecipitation with nonspecific mouse IgG was performed on the Etoposide (Eto.) sample as a technical control for the procedure. Both in the presence of ZFP36 or Etoposide (Eto.) RIP1 was associated with caspase8, while this was less evident in the empty vector population (E.V.). Higher levels of FADD were associated to caspase8 in the empty vector population, as opposite to the ZFP36 and Etoposide (Eto.) samples. Input controls are represented on the right panel. C, D) Image J software provided a relative quantification of the levels of RIP1 and FADD associated to caspase8.