Immunoblotting Analysis of yTdp1 Protein

Immunoblotting was performed as described in Gajewski et al.^{21 (link)}. Briefly, we used anti-yTdp1^{8 (link)}, anti-Histone H3 (Abcam), and anti-GAPDH (Gentex) antibodies. Transformed yeast cells with the indicated vectors were grown in selective media supplemented with 2% dextrose the first night, then 1/100 diluted into selective media supplemented with 2% raffinose. Raffinose cultures were induced for 6 h with 2% galactose, corrected to the same OD₆₀₀ and lysed with acid-washed glass beads (Sigma) at 4 °C in 50 mM Tris pH 8.0, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF, 10% glycerol and Complete EDTA-free Protease Inhibitor (Roche). Lysate protein concentrations were determined by Bradford-assay (Bio Rad). Lysates were boiled in SDS buffer for 10 min and samples were loaded onto a 10% Bis–Tris PAGE in SDS buffer, blotted onto PVDF and immunostained with anti-yTdp1, stripped (62.5 mM Tris, 2% SDS and 0.8% β-mercaptoethanol) and reprobed with anti-GAPDH or anti-Histone H3 antibodies. Immunostaining was visualized by chemiluminescence using a G:Box imager and the accompanied GeneSynV1.6.1.0 software (Syngene). For presentation, orientation, and intensities (by correcting brightness and contrast) of whole gel images were modified in Adobe Photoshop before cropping and figures were finalized (adjusted in size) in Adobe Illustrator.