Immunoprecipitation experiments
Flag-immunoprecipitation followed by western blotting was used to analyze the modification status of the precipitated proteins and their mutant versions. Full-length human histone H3.1 and HPF1 cDNAs were cloned into the pDONR221 vector (Thermo Fisher Scientific). Point mutations were produced in pDONR-H3.1 and pDONR-HPF1 using QuikChange Lightning site-directed mutagenesis kit (Agilent). Mammalian expression constructs expressed H3.1 proteins with the C-terminal 3xFlag tag, and HPF1 proteins with N-terminal Flag tag. Wild-type proteins and their mutant versions were expressed in 293T cells. The cells were plated, cultured overnight, and transfected using Polyfect (QIAGEN) with an empty vector or a plasmid expressing the Flag-tagged protein of interest for 24 hr essentially as described (Palazzo et al., 2018). The cell lysates were obtained the same as for the western blotting. Protein concentrations were analyzed by Bradford Protein Assay (Bio-Rad), normalized, and then, the cell lysates were incubated with anti-Flag M2 agarose-conjugated mouse monoclonal antibody (Sigma-Aldrich) for 1 hr while rotating at 4°C. Beads were washed several times with Triton X-100 lysis buffer and eluted with NuPAGE LDS sample buffer (Invitrogen). The samples were then analyzed by Western Blotting as described above.