Anti myc clone 9e10

Single colonies of yeast strains expressing Rad52-FLAG or HsRAD52-FLAG from genomically integrated fusion genes, and Rad51-MYC, Rad52-MYC or HsRAD52-MYC from fusion genes on plasmids containing the appropriate combination of tagged alleles were used to inoculate cultures of synthetic complete medium lacking uracil and grown overnight. The overnight cultures were used to inoculate YPD cultures that were grown to mid-log at 30°C. Whole cell extracts were prepared by silica bead disruption and pre-cleared by the addition of pre-blocked protein A/G agarose beads (Pierce Thermo-Fisher Scientific, Waltham, MA, USA). Pre-cleared extracts were mixed with either anti-FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO, USA) or anti-MYC clone 9E10 (Santa Cruz Biotechnology, Dallas, TX, USA), followed by the addition of pre-blocked protein A/G beads. Immunoprecipitates were eluted from the beads and loaded onto acrylamide gels, the proteins separated by electrophoresis and then electrophoretically transferred to nylon. Protein signals were revealed using either a HRP-conjugated anti-FLAG antibody, or the mouse anti-MYC clone 9E10 primary antibody and HRP-conjugated goat anti-mouse secondary antibody as described previously.

The following antibodies were used for Western blots (WB), immunofluorescence (IF), and Filter trap (FT): anti‐DYKDDDDK(FLAG) Tag (Cell Signaling #2368S) WB 1/1,000, anti‐myc clone 9E10 (Santa Cruz) WB 1/1,000, IF 1/100, anti‐HA Tag clone 3F10 (Roche) WB 1/1,000, IF 1/100, anti‐GFP(B‐2) (Santa Cruz) WB 1/500, FT 1/500, anti‐beta‐actin (Sigma‐Aldrich) WB 1/1,000, FT 1/1,000, anti‐EXOSC10 (ATLAS ANTIBODIES) WB 1/500, IF 1/2,000, anti‐DIS3 (Proteintech) WB1/2,000, anti‐DIS3L (Santa Cruz) WB 1/500, anti‐GAPDH (Proteintech) WB 1/20,000, anti‐HDAC1 (Proteintech) 1/1,000, anti‐PR (Proteintech) WB 1/500, FT 1/500, anti‐GR (Proteintech) WB 1/1,000, FT 1/1,000.