Anti pgk1 antibody 22c5d8

Rad53-FLAG expressing cells were grown to 5 × 10⁶ cells/ml in YPAD, synchronized with α-factor for 2 h, washed with YPAD and released in YPAD containing 0.2 M HU (Sigma-Aldrich). Samples were collected at 0, 20, 40, 60, 80, 100 and 120 min after release. Chl1-FLAG and Chl1^K48R-FLAG expressing cells were grown to 5 × 10⁶ cells/ml in YPAD. Chl1-Myc-AID expressing cells were grown to 5 × 10⁶ cells/ml in YPAD and synchronized with α-factor for 2 h. Whole cell extracts were prepared by TCA precipitation and analyzed by SDS–PAGE. Western blotting was performed using an anti-Flag antibody (clone M2; Sigma-Aldrich) or anti-Myc antibody (Santa Cruz [9E10: sc-40]). An anti-PGK1 antibody (22C5D8; Invitrogen) was used to control protein loading.

The cells collected from indicated times were then treated with sodium hydroxide for protein extraction, as described previously [44 (link)]. Protein samples were loaded on to an 8% or 10% SDS-PAGE gel for protein electrophoresis and then transferred to a PDVF membrane (Millipore) for Western blot analysis. Anti-FLAG polyclonal antibody M2 (Sigma), anti-Mcm2 polyclonal antibody N-19 (Santa Cruz), anti-Mcm4 polyclonal antibody yC-19 (Santa Cruz), anti-Mcm7 polyclonal antibody yN-19 (Santa Cruz), and anti-Elm1 polyclonal antibody y-640 (Santa Cruz) were used to detect 3Flag-Lrg1, Mcm2, Mcm4, Mcm7, and Elm1, respectively. The monoclonal anti-Pgk1 antibody 22C5D8 (Invitrogen) was used to detect Pgk1, as the loading control, since Pgk1 is reported to be a very stable protein based on its half-life [45 (link)]. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore). Signal intensities were quantified by means of Image Quant (GE Healthcare).