HA-Immunoprecipitation of Transfected MCM Proteins

HEK293T were transiently transfected with pMSCV-FLAG-HA-MCM8, -MCM8 L387E, -MCM9, -MCM9 M45E or -GFP control (Transporter 5, Polysciences 26008-5) and harvested 3 days later for HA-immunoprecipitation^{33 (link)}. Briefly, cell pellets were resuspended in mammalian cell lysis buffer (MCLB) (50 mM Tris-HCl pH 7.5, 1% IGEPAL CA-630) supplemented with 150 mM NaCl, a protease inhibitor (Goldbio, GB-331) and phosphatase inhibitor cocktail (Sigma, 4906837001). Following incubation for 30 minutes at 4 °C, extracts were cleared by centrifugation and supernatants collected. The remaining pellets were subjected to a second round of extraction by resuspending them in MCLB supplemented with 500 mM NaCl and protease (Goldbio, GB-331) and phosphatase inhibitor cocktail (Sigma, 4906837001), and incubating them for an additional hour at 4 °C. After clearing these extracts by centrifugation, the NaCl concentration of the collected supernatants was adjusted to 150 mM with MCLB and combined with the supernatants from the first round of extraction. Combined lysates were then incubated with anti-HA agarose beads (Sigma, A2095) for 4 hours at 4 °C. After incubation, beads were washed four times in MCLB buffer supplemented with 150 mM NaCl, and then boiled in LDS (lithium dodecyl sulfate) sample buffer (Thermo Fisher, NP0008) supplemented with β-mercaptoethanol to elute the bound proteins.