CERKL interacts with components of the translation machinery and with mRNAs.

A) HEK-293T cells overexpressing Flag-tagged GFP, CERKL- WT or CERKL-C125W were lysed and Flag was immunoprecipitated as described in Materials and Methods, analyzed by SDS-PAGE in 8% (left panel) and 20% (right panel) polyacrylamide gels and silver-stained. Proteins were characterized by mass spectrometry (see Materials and Methods). The positions of the proteins are indicated by arrows, from up to down as follows: eIF3B (1), CERKL (2), alpha-tubulin (3), ß-tubulin (4) (8%polyacrylamide gel), PABP (5), HSP70 (6), CERKL (7) and RPS3 (8) (20% polyacrylamide gel, WT lane) and GFP (9) (20% polyacrylamide gel, GFP lane). B) Flag immunoprecipitations (FLAG IP) of protein homogenates from HEK 293T cells transfected with CERKL-Flag (WT), CERKL-C125W-Flag (CW) or, as a control, GFP-Flag (GFP). The position of CERKL and GFP, detected with anti-Flag, is shown. Specific antibodies were used to detect eIF3B, PABP, HSP70 and RPS3. C) EMSA of CERKL protein incubated at the indicated concentrations with biotinylated mRNAs from human retina. BSA (CONTROL) and His-MBP (0) were used as negative controls. Addition of an excess of non-biotinylated probes reduced the intensity of the shifted bands (COMPETITION). Arrowheads indicate the position of the shifted bands. D) Two CERKL proteins moieties, Nter (amino acids 1–256) and Cter (amino acids 252–532) and the two negative controls from C were analyzed by EMSA using biotinylated mRNAs from COS-7 cells. Arrowhead indicates the position of the shifted band. E) Flag-tagged CERKL-WT or GFP proteins transiently expressed in HEK 293T cells were purified by immunoprecipitation, eluted with Flag peptide and then incubated with m7-GTP bound to Sepharose 4B beads (M7-GTP). Proteins bound to these beads were analyzed by Western blot with anti-Flag. As control, unconjugated Sepharose 4B beads (Seph) were used. Only CERKL-WT-Flag, but not GFP-Flag, binds to m7-GTP-Sepharose.