Monoclonal anti gfp

Chromatin immunoprecipitation analysis (ChIP), gene expression analysis by RT-PCR, protein co-immunoprecipitation assays and western blot analysis were performed as previously described (Gomar-Alba et al., 2017 (link)). In ChIP assays, Dynabeads Protein G magnetic beads were incubated with HA-probe (F-7) antibody (Santa Cruz Biotechnology; SC-7392) or monoclonal anti-GFP (Roche Diagnostics; 11814460001). The primary antibodies used in the western blot analysis were monoclonal anti-HA peroxidase 3F10 antibody (Roche Diagnostics; 12013819001) diluted 1:5000, monoclonal anti-GFP (Roche Diagnostics; 11814460001) diluted 1:5000 and monoclonal anti Cdc2 p34 (PSTAIRE; Santa Cruz Biotechnology; SC-53) diluted 1:2000. Blots were developed using anti-mouse IgG and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:20,000; Thermo Fisher Scientific; 170-6516 and 31460, respectively) and Supersignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific). Bands were quantified using an ImageQuant LAS 4000mini Biomolecular Imager (GE Healthcare).

Total cell extracts were prepared by incubation in lysis buffer containing 50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 150 mmol/l KCl, 1 mmol/l dithiothreitol, 5 mmol/l EDTA, pH 8.0, 1% Nonidet P-40 plus a mix of protease and phosphatase inhibitors (Roche Diagnostic) and resolved by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore) and incubated with the primary antibodies followed by an anti-immunoglobulin-G-horseradish peroxidase antibody (Bio-Rad). Specific proteins were detected by enhanced chemiluminescence (ECL) (Amersham). Immunoblotting was performed with: mouse monoclonal anti-Poly (ADP-ribose) polymerase (PARP) (cleaved form, BD Pharmingen), rabbit polyclonal anti-p53 (FL393) mouse monoclonal anti-p53 (DO1) (both from Santa Cruz Biotechnology), monoclonal anti-GFP (Roche Diagnostic), polyclonal anti-Bax (N20, Santa Cruz Biotechnology), monoclonal anti-phospho-Histone H2A.X (Ser139) (Millipore) (a kind gift from S. Soddu) and monoclonal anti-β-actin (Calbiochem) antibodies.

All yeast culture and genetic techniques were performed as previously described [24 (link)]. S. cerevisiae strains used in this study are listed in Table S1 in the Supplementary Materials. Yeast strains were constructed using a PCR-based one-step gene disruption technique [30 (link)]. Various deletion constructs were constructed on centromeric shuttle plasmids and transformed in to yeast for analysis [31 (link)]. The GAL1 promoter was induced for full-length Fus2p and Cla4p expression using 2% galactose and for Fus2p truncations with 2% galactose + 0.2% glucose. To induce mating differentiation, 10 µg/mL α-factor was used. Plasmids used in this study are listed in Table S2 in the Supplementary Materials. Point mutations of FUS2 or CLA4 were generated by PCR-based site-directed mutagenesis. Proteins were prepared by TCA precipitation, resolved on gels with or without Phos-tag (Wako Chemicals, Richmond, VA, USA), and detected on Western blots with monoclonal anti-GFP (Roche, South San Francisco, CA, USA), monoclonal anti-c-Myc (Sigma, St. Louis, MO, USA), monoclonal-HA (Thermo Scientific, Waltham, MA, USA) and monoclonal anti-MBP (Millipore, Burlington, MA USA). Band intensity was quantified using a G:BOX imaging system (Syngene, Frederick, MD, USA).