Image gauge

Phospho-specific antibodies against phosphorylated Mrc1-Thr⁶⁴⁵, Cds1-Thr¹¹, and Rad9-Thr⁴¹² were generated using chemically synthesized phosphopeptides (13 (link),15 (link)). Rad9 and Cds1 were tagged with hemagglutinin (HA) epitope and immunoprecipitated (IPed) from whole cell lysates made by glass-bead method in the buffer containing 50 mM HEPES/NaOH, pH 7.6, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM pyrophosphate, 50 mM NaF, 60 mM β-glycerophosphate, 0.1% Nonidet P-40, and protease inhibitors. After separation by SDS PAGE, samples were transferred to a nitrocellulose membrane. The membrane was briefly probed with anti-HA antibody (Santa Cruz Biotechnology) to confirm the loading. Immunobloting signal was detected by electrochemiluminescence using Image Reader LAS-3000 (Fuji Film). Band intensities were quantitated by ImageGauge (Fuji Film). The same blots were stripped and reprobed with phospho-specific antibodies against Rad9-Thr⁴¹² or Cds1-Thr¹¹ for 3 h at room temperature or overnight at 4 C. Phosphorylation of Mrc1-Thr⁶⁴⁵ was directly examined in whole cell lysates prepared by trichloroacetic acid (TCA) method (15 (link)). The membranes were stripped and blotted with polyclonal antibodies against Mrc1 as the loading control. Phosphorylation of Chk1 by Rad3 was assessed by using the established mobility-shift method (37 (link)).

Fluorescence anisotropy and electrophoretic mobility shift assays (EMSA) using purified recombinant POS-1 (80–180), MEX-3 (45–205) and MEX-5 (236–350) were done as described in Farley et al. 2008 (link), Pagano et al. 2009 (link) and Pagano et al. 2007 (link), respectively. All RNA oligonucleotides used in this study were chemically synthesized and fluorescently labeled at the 3′ end with fluorescein amidite (FAM) by Integrated DNA Technologies (IDT).
Competition assays are set up similar to the EMSA assays. 550 nM of POS-1 (80–180) or 450 nM MEX-5 (236–350) was added to the RNA equilibration buffer to obtain a 70% RNA bound complex. Then the corresponding competing protein was titrated to the reaction mixture at varying concentrations. After 3 hours of equilibration, the reaction mixture was run on a 5% native polyacrylamide gel in 1X TB for 3 hours, at 120V. Quantifications were done by determining the pixel intensity of the RNA species bound by protein relative to the pixel intensity of total RNA species to give the fraction bound of RNA. The pixel intensities of each band were determined and background corrected by using Image Gauge (Fujifilm, Tokyo, Japan).

Electrophoretic mobility shift assays using purified recombinant MBP-tagged GLD-1 and mutant variants were performed essentially as described (Farley and Ryder, 2012 (link)). The target RNA sequence (RNA: 5’-UUUUU CUUAU UCUAG ACUAA UAUUG UAAGC U-3’-Fl), comprising a fragment of the glp-1 3’UTR previously shown to bind to GLD-1 (Farley and Ryder, 2012 (link)), was chemically synthesized and labeled at the 3énd with fluorescein amidite (FAM) by Integrated DNA Technologies (IDT, Coralville, IA). Varying concentrations of purified GLD-1, GLD-1 (G248R) and GLD-1 (G250R) were equilibrated with 3 nM of labeled RNA in equilibration buffer (0.01% IGEPAL, 0.01 mg/ml tRNA, 50 mM Tris, pH 8.0, 100 mM NaCl, 2mM DTT) for 3 hours. After equilibration, bromocresol loading dye was added, the reaction mixture was loaded onto a 5% native polyacrylamide gel in 1X TBE, and allowed to run for 2 hours at 120 volts. The gels were quantified by determining the pixel intensity of the RNA bound species and the total of all RNA species to give the fraction bound of RNA relative to the background using Image Gauge (Fujifilm, Tokyo, Japan). The graph of fraction bound against the protein concentration was then fit to the Hill equation to determine the apparent binding affinity and the Hill coefficient.