(A) Retesting of mutants for impaired growth on MTX-containing medium.

<p>Selected candidates from a rPCA screen against a query strain harboring the fusion proteins Cia2:: F[1,2] and Mms19:: F[3], were backcrossed to a wild type strain, sporulated, and subjected to tetrad dissection. Ten-fold serial dilutions of the indicated haploid spores were spotted on a rich medium lacking (control), or supplemented with 200 µg/ml MTX. Cells were incubated at 30°C, 34°C, and 37°C to identify the semi-permissive temperature of each temperature sensitive (Ts) mutant (30°C and 34°C are shown). A wild-type strain was used as a positive control. Δ<i>apn1</i>, which plays a specific role in DHFR biogenesis, was used as a negative control for growth on MTX. <b>(B) Reduced association between Mms19 and Cia2 in yeast cells depleted for Cia1, a member of the CIA complex. </b><i><u>Left</u></i>-Western blot confirming that the expression levels of Cia1 regulated by a galactose-inducible promoter (<i>GAL1</i>), are abolished in glucose containing media. The expression of <i>GAL1</i>-<i>CIA1</i>-FLAG was induced by growing the cells in 2% galactose (Gal) for 3 hours (t-0). Then, 2% glucose (Glu) was added to the media to shut-off the expression of <i>CIA1</i>, and samples were collected at the indicated time points for immunoblotting with anti-FLAG, and anti carboxypeptidase-Y (CPY) ([IB]:α-CPY) (loading control). Quantitation of the band representing the Cia1-FLAG relative to the CPY loading control is shown in the graph. <i><u>Right</u></i>- Protein extracts were prepared from yeast strains carrying a galactose-inducible <i>CIA1</i>, and the combinations of Cia2-Myc and Mms19-GFP grown in 2% glucose for 24 hours (to deplete the levels of Cia1). After immunoprecipitation (IP) with anti-myc antibody, whole cell extracts (WCE), and immunocomplexes (IP:α-Myc) were separated by SDS-PAGE, and immunoblotted with anti-GFP and anti-Myc antibodies. Immunoblotting with anti-phosphoglycerate kinase1 (PGK1) antibody was used as a loading control. A strain carrying Mms19-GFP served as a negative control. Quantitation of the bands representing the precipitated Mms19-GFP relative to the total amount of Mms19 (WCE) is shown in the graph. <b>(C) siRNA-mediated depletion of CIAO1 in HeLa cells affected the endogenous levels of hCia2, and its association with hMms19.</b> Protein extracts were prepared from HeLa cells following the siRNA mediated knock down of CIAO1 (siCIAO1), or a non-targeting siRNA control (siCONTROL). WCE and immunoprecipitates (IP:α-Mms19) were separated by SDS-PAGE. hCia2, hMms19 and CIAO1 were immunostained using their specific antibodies. α-tubulin was used as a loading control. Quantitation of the band representing the hCia2 in the WCE relative to tubulin loading control is shown in the <i>left</i> graph. Quantitation of the bands representing the precipitated hCia2 relative to the amount of hCia2 in the WCE, and immunoprecipitated Mms19 is shown in <i>right</i> graph.