Figure 5
OFD1 cooperates with Bicc1 to control the translation of specific mRNAs. (a) Co-IP experiments demonstrate that the IP3 buffer destroys OFD1/eIFs interactions, which are preserved using the Co-IP buffer. (b) Real-Time PCR of the OFD1-bound mRNA shows that Net1, Vcl, Gh, Gdi2 and Vps39 are not enriched after OFD1 IP (black bars) compared to Control (eIF4E-IP white bars). (c) Silencing of Bicc1 results in stronger eIF3B/OFD1 affinity. Similar results are observed when OFD1 is silenced and eIF3B/Bicc1 interaction is analyzed. (d) Real-Time PCR of the Bicc1-bound mRNA shows that mRNAs target are enriched after Bicc1 IP (grey bars) compared to Control (eIF4E-IP white bars). (e) RNA binding experiments are performed in OFD1- and Bicc1-silenced cells. In the absence of OFD1 the binding of target mRNAs to eIF4E (black bars) is more efficient compared to controls (white bars); while silencing of Bicc1 results in decreased mRNA enrichment (grey bars). (f) IF with an antibody against Bicc1 (green) shows that Bicc1 colocalizes with γtubulin (red) at the centrosome and that the amount of centrosomal Bicc1 increases in OFD1-silenced cells (white arrows). Representative superresolution images are reported and a magnification of the centrosome is shown in the dotted white box. IF in Bicc1-silenced cells is reported as control for antibody specificity. Bicc1 localization at the centrosome was quantified by ImageJ and reported in a graph on the right. Main fluorescence intensity was calculated in the centrosomal area and reported in white for controls (C) and in black for OFD1-silenced cells (siOFD1). Data are presented as the mean + SEM. ns p-value > 0.05; *p-value < 0.05; ***p-value < 0.01 ****p-value < 0.005. Representative images were taken at the same contrast and reported. Bar = 5 μm.
