Figure 1
OFD1 interacts with the translational machinery. (a) OFD1 interactome. The putative OFD1 partners were clusterized in functional classes, which are identified by a color code. The majority of interactors are involved in the control of the cellular protein content (different shades of orange and red). (b) OFD1 interacts with components of the PIC and the eIF4F complex. Co-IP experiments performed on total lysates (TL) from HEK293 cells. (Upper panels) eIF3B and eIF3G were immunoprecipitated and OFD1 was detected by WB. (Lower panels) OFD1 was immunoprecipitated and different subunits of the PIC and of the eIF4F complex (eIF3B, eIF3G, eIF4G, eIF4E) were detected by WB. IgG were used as control. Holocytochrome-c type-synthase (HCCS) and Citrato synthase (CS) were used as negative controls. All lanes are from the same blot. However some of the lanes were not adjacent and are separated by a space. (c) Co-IP experiments demonstrate that eIF4E binds other PIC and eIF4F components (eIF3B, eIF4G and S6) in OFD1 silenced cells. Lower panel in c reports the effect of OFD1 silencing at the protein level. (d) OFD1 directly binds eIF4E. An eIF4E binding site (eIF4E-BS, Aa in red) is recognized in the OFD1 protein and conserved in OFD1 vertebrate homologs. Aa mutagenized within the eIF4E-BS are depicted in green. Co-Ip experiments demonstrated that the mutated constructs show a lower affinity for eIF4E (right panel). βtubulin was used as loading control. TLs are reported on the left. The eIF4E/OFD1 ratio was calculated and the fold change was reported below the panels as the mean ± standard error of the mean (SEM). (e) eIF4E mediates the interaction of OFD1 with eIFs. OFD1 was immunoprecipitated in control and eIF4E-silenced HEK293 cells and eIF4G, eIF3B and eIF4E were detected by WB. IgG were used as loading control.
