Figure S3. In vitro analysis Pch2–(ORC) origin recognition complex assembly.

(A) Size exclusion chromatography of the ORC (His–Orc1, Orc2-6) purified from insect cells. Coomassie Brilliant Blue (CBB) staining of peak fractions (dotted line) run on SDS–PAGE gel. AU, arbitrary units. (B) Western blot of the purified ORC (His–Orc1, Orc2-6) using α-ORC. (C) MS-analysis of the insect cell–purified ORC. (D) CBB staining of the purified ORC (His–Orc1, insect cells) and the budding yeast ORC (CBP–Orc1). (E) Treatment of the purified ORC (His–Orc1, insect cells) with λ-phosphatase, at 4°C or room temperature. CBB staining. # indicates phosphorylated Orc6. (F) Amylose-based pulldown of the dephosphorylated and untreated ORC (His-Orc1, insect cells), with His–MBP–Pch2 or MBP–His; Coomassie Brilliant Blue (CBB) staining. # indicates phosphorylated Orc6. (G) Calmodulin-based pulldown of the ORC (Orc1-6) purified from budding yeast, with His–MBP–Pch2 and MBP–His. Coomassie Brilliant Blue (CBB) staining. (H) Calmodulin-based pulldown of the ORC (Orc1-6) purified from budding yeast, with His–MBP–Pch2 and MBP–His, in the presence or absence of MgCl₂ and ADP/ATP (and ATP analogs). Coomassie Brilliant Blue (CBB) staining.