Nucleoporins facilitate ORC loading onto chromatin
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Raw images for western blots found within the paper: Nucleoporins facilitate ORC loading onto chromatin
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The presence of ORC2 in the elute was confirmed prior to conducting mass spectrometry by SDS-PAGE followed by a Western blot for ORC2 using anti-ORC2 antibody. Briefly, samples were boiled and loaded onto a 4–15% Mini-PROTEAN TGX Stain-Free Gel (Biorad). After electrophoresis, the gel was activated and imaged using a BioRad ChemiDoc™ MP Imaging System following manufacturer recommendations. Protein was transferred to a low fluorescence PVDF membrane using a Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked with 5% milk in TBST (140 mM NaCl, 2.5 mM KCl, 50 mM Tris HCl pH 7.4, 0.1% Tween 20). Blots were incubated with either anti-ORC2 antibody at 1:1000, anti-Elys antibody at 1:250, or HRP anti-Histone H3 antibody (abcam #ab21054) at 1:1000 overnight at 4°C. After primary antibody incubation, blots were washed, incubated secondary HRP antibody (Jackson Labs 711-035-150), washed once more and imaged. For the quantification shown in Figure S4B, signal for either Elys or ORC2 was normalized to H3 for each depletion for three biological replicates.