Fig 1: SDOS localizes on the ER and associates with actively translating polyribosomes. (A) Subcellular fractionation of HeLa cells into mitochondrial (Mito), cytosolic (Cyto) and microsomal (ER) fractions following 24 h induction of SDOS-eGFP. Total lysate and fractions were analyzed by WB with indicated antibodies. BiP is used as a ER marker, GAPDH as a cytosolic marker and VDAC1 as a mitochondrial marker. (B) WB analysis of TRAP1-IP from the ER fraction with SDOS, TRAP1 antibodies. Vinculin is used as internal control. (C) Ribosomal purification from HCT116 cells and HCT116 cells transfected with SDOS-myc, followed by immunoblot with ß-ACTIN (as a cytosolic marker), ßF1ATPase (as a mitochondrial marker), RPL3 (as a ribosomal marker) and SDOS antibodies. (D) Polysome profiling absorbance, measured at 254 nm, indicates the sedimentation of the particles: fractions 1 and 2 free cytosolic proteins or light complexes; fractions from 3 to 7 ribosomal subunits (60S, 40S) and monomer (80S); fractions from 8 to 12 polysomes. Proteins from each fraction were analyzed by WB with the indicated antibodies. rpL3 was used as a ribosome marker, eIF4B and eIF4E were used as markers of translation initiation complex. (E) Representative image of PLA showing the interaction of SDOS-eGFP with phosphorylated (active) ribosomal protein S6 (phospho-rpS6) in HeLa cells following 24 h induction of SDOS-eGFP by using GFP and phospho-rpS6 antibodies. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Cycloheximide treatment (200 µg/ml, 1 h) was used to confirm the causal role of active protein synthesis on the binding.
Fig 2: SDOS binds and regulates translation of mRNAs encoding for ciliary components. (A) RT-qPCR of Cc2D2a, Kif7 and Tmem107 mRNAs from HeLa cells upon 24 h of SDOS-eGFP or eGFP induction (ßActin used as the internal control). Data are expressed as mean ± S.E.M. from four independent experiments with technical triplicate each. Numbers above bars indicate the statistical significance (P-value), based on one-sample t-test. Red line indicates expression level of the relative eGFP control. (B) Representative results of RT-qPCR from RIP independent experiments to validate iCLIP data. RNA enrichment was normalized to a spike-in control (red line). Actin was used as negative control showing no enrichment. (C and D) WB analysis of HeLa cell extracts following SDOS-eGFP or eGFP (24 h) and sh-GFP or sh-SDOS (48 h) induction (C), with relative densitometric analysis (D), calculated by assuming protein levels of the control equal 1. The P-value in the graph indicate the statistical significance based on one-sample t-test (n = 3).
Fig 3: SDOS expression alters cilium formation. (A) Representative image of the primary cilium (red) and its length in HeLa cells upon overexpression of SDOS-eGFP or the respective eGFP control. Cilium formation has been induced by serum starvation (48 h), then cells have been fixed, permeabilized and hybridized with Acetyl-Tubulin as a primary cilium marker. Nuclei are stained with DAPI (blue). Scale bar: 15 µM. (B) Representative image of the primary cilium (green) and its length in HeLa cells upon induction of SDOS-directed shRNA and the respective shGFP control. Cilium formation has been induced by serum starvation (48 h), then cells have been fixed, permeabilized and hybridized with Acetyl-Tubulin as a primary cilium marker. Nuclei are stained with DAPI (blu). Scale bar: 15 µM. (C) Length of primary cilia measured by confocal microscopy analysis of Acetyl-Tubulin-stained HeLa cells upon SDOS overexpression (SDOS-eGFP, n = 146) or silencing (shSDOS, n = 110) and relative eGFP (n = 165) and shGFP (n = 168) controls. Numbers indicate the statistical significance (P-value) based on the Student’s t-test. (D) Bar graph representing the percentage of ciliated HeLa cells upon SDOS silencing (shSDOS) or overexpression (SDOS-GFP) compared to the respective shGFP and eGFP controls.
Fig 4: SDOS overexpression represses translation of specific mRNAs. (A) Autoradiography of total lysates from cells labeled with 35S Met/35S Cys (left) and relative densitometric band intensities (right), calculated by assuming protein levels of the control equal 1, following 24 h induction of eGFP/SDOS-eGFP or 48 h of shGFP/shSDOS in Hela FITR or transfection of SDOS-targeting siRNA and non-targeting control siRNA in HCT116 cells. The P-value in the graph indicate the statistical significance based on one-sample t-test (n = 3). (B) WB analysis of total lysates following puromycin treatment (1 µg/ml, 15 min) (left) and relative densitometric band intensities (right), calculated by assuming protein levels of the control equal 1. The P-value in the graph indicate the statistical significance based on one-sample t-test (n = 3).
Fig 5: SDOS and TRAP1 are protein partners. (A) Volcano plot showing SDOS (left) and TRAP1 (right) protein partners identified by MS (dark-red = significant, light blue = intermediate, light gray = not significant). Relevant protein-partners (RPS28, SDOS, TRAP1, TP53BP1) are highlighted. (B) Silver staining showing the pattern of SDOS-eGFP and TRAP1-eGFP IPs (specific band indicated with asterisk). (C) Co-immunoprecipitation of TRAP1 and SDOS from HeLa cells following induction of TRAP1-eGFP and eGFP for 24 h. Total lysates were immunoprecipitated with GFP-Trap_MA magnetic agarose beads and analyzed by WB with the indicated antibodies. (D) Representative image of PLA showing the interaction of SDOS with TRAP1 in HCT116 cells. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Negative control has been obtained by hybridizing cells with TRAP1 antibody only. (E) Representative image of PLA showing the interaction of SDOS with rpS28 in HeLa cells, following induction of SDOS-eGFP or unfused eGFP (negative control) for 24 h and hybridization with anti-GFP and anti-rpS28 antibodies. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). (F) Computational prediction of SDOS protein partners network using STRING database (https://string-db.org). Interactions include direct (physical) and indirect (functional) associations. Light-blue edge: known interaction from curated databases; purple edge: experimentally determined interaction; green edge: textmining; black edge: co-expression; dark-blue edge: protein homology.
Supplier Page from MilliporeSigma for Anti-NUDT16L1 antibody produced in rabbit