Anti lexa

For Western blot analysis, yeast cells (O.D₆₀₀ = 1.5) were collected by centrifugation at 3000 rpm in a table-top centrifuge for 5 min. The cell pellets were resuspended in 100 μl of SDS-buffer (50 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 5 min. After the lysates were cleared by centrifugation at 12,000 rpm for 10 min, soluble proteins were resolved by SDS–PAGE and transferred to PVDF membrane (Millipore). The membranes were incubated with appropriate antibodies (anti-HA, anti-LexA, anti -GFP and anti-TAP antibodies, Santa Cruz) in TBST buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and proteins were detected by the enhanced chemiluminescence (ECL) system. For IP, yeast cells were disrupted by vortexing with acid-washed glass beads in ice cold NP40 buffer (1% NP40, 150 mM NaCl, 50 mM Tris–HCl, pH 8.0) containing protease and phosphatase inhibitors. The cell lysates were incubated with appropriate antibodies at 4 °C for 3 h and further incubated with protein A/G-conjugated agarose beads at 4 °C for 1 h. The precipitated agarose beads were washed three times with ice cold NP40 buffer containing protease and phosphatase inhibitor cocktails (Sigma P8215 and Sigma P0044, respectively) and boiled in 50 μl of SDS–PAGE buffer. The resulting proteins were analyzed by Western blot using appropriate antibodies.

Denaturing whole-cell extracts were prepared in10% trichloroacetic acid by agitation with glass beads. Proteins were solubilized in Laemmli sample buffer, separated by SDS-PAGE and analyzed by western blotting. Antibodies were goat polyclonal anti-LexA (1:2000, Santa Cruz), rat polyclonal anti-α-tubulin (1:5000, Bio-rad), mouse monoclonal anti-Flag M2 (1:2000, Sigma) HRP-conjugated mouse monoclonal anti-Flag M2 (1:2000, Sigma), mouse monoclonal anti-MBP (1:2000, NEB), rabbit polyclonal anti-Spo11 (1:1000, this laboratory). Secondary antibodies were used at 1:5000 dilution: IRDye 800CW goat anti-mouse IgG (LI-COR), HRP-conjugated goat anti-mouse IgG (Bio-Rad), HRP-conjugated goat anti-rabbit IgG (Bio-Rad), HRP-conjugated donkey anti-goat IgG (Santa Cruz), HRP-conjugated donkey anti-rat IgG (Abcam).