Figure 3
OFD1 is present in polysomal fractions and regulates the translation of specific targets in the kidney. (a) OFD1 coimmunoprecipitates with eIF3B in vivo in polysomes. We immunoprecipitated the OFD1 protein in TL (first lane) and in polysomal fractions (P, second lane) both in HEK293 cells (top panel) and in wt kidney extracts (P8) (lower panels) and the OFD1 protein was detected by WB. In TL the abundance of immunoprecipitated OFD1 probably reflects the localization of the protein in different cell compartments compared to the P fraction. Co-IP experiments confirmed that the interaction between OFD1 and eIF3B occurs also in vivo in polysomal fractions. eIF3B and rpS6 were immunoprecipitated from P fractions. Bands appeared enriched. These markers were used to demonstrate specificity of the P fractions. GAPDH was detected by immunoprecipitation in TL from kidneys and was absent in P and was used as a control for specificity. HCCS was used as a negative control both for the specificity of polysomal fractions and of the Co-IP. All lanes are from the same blot. However some of the lanes were not adjacent and are separated by a space. (b) Polysomal profiles observed in kidneys from Ofd1-IND mutants at P8 (upper panels) and P20 (lower panels). The peaks show the absorbance (Abs 254 nm) measured in the collected fractions. The polysomal (P) and subpolysomal (SP) areas are indicated. The polysomal fractions were obtained from two independent experiments for each sample. (c) Analysis of the polysomal profile. The analysis was performed on renal extracts from Ofd1-IND mice calculating the ratio between Polysomal RNA (P-RNA) and subpolysomal RNA (SP-RNA) in Controls (white bars) and Ofd1-IND mice (black bars). The RNA was obtained from kidneys of Ofd1-IND mutants and Controls at P8 (precystic stage) and at P20 (cystic stage). Data are presented as the mean ± SEM. Student’s t-test was used to calculate the p-value. **p-value < 0,05 at P20.
