Gapdh

ChIP was performed according to the ChIP-IT kit protocol (Active Motif). Control IgG, RNA Pol II, and Ezh2 antibodies were from Active Motif. Control ChIP primers toward glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Active Motif. Primer sequences included forward (-7885) 5′-GCAACAGTTAGCATCATGGGC-3′, reverse (-7885) 5′-TGGGGTTGTAGGCTTTCCTG-3′, forward (-3389) 5′-CAGGCCATGGCTGTTTGATG-3′, reverse (-3389) 5′-CAGGTGCATGGGGTACAGAG-3′, forward (TSS) 5′-CGGAAAGGGGTTTGTTGCTGG-3′, reverse (TSS) 5′-GGGACGCCCCTGTCCTTTAC-3′.

HeLa Kyoto cells were grown in 96-well plates for 24 h in complete medium at 37°C in 5% CO₂. Luciferase assays were performed with the following promoter luminescent reporter gene constructs: SEC23A, SEC23B, SEC24B, SEC24C, SEC24D, and GAPDH (Active Motif). All promotor constructs were obtained from the Active Motif LightSwitch Promoter Reporter GoClone Collection (Trinklein et al., 2003 (link)). Cells were cotransfected with 50 ng GoClone plasmid DNA per well and with 10 ng of either turboGFP-tagged RNF11 or GFP empty vector according to the manufacturer’s instructions (Active Motif). Plasmid DNA expression time was 24 h for all experiments. Luciferase reporter signal was measured with the LightSwitch Luciferase Assay System (Active Motif) on a SpectraMax L luminometer according to the manufacturer’s instructions. Relative luminescence units of GFP-expressing cells were normalized to RNF11-GFP–expressing cells. To achieve this, in parallel to the 96-well plate for the luciferase assay, a 96-well glass-bottom plate was assessed on a standard wide-field fluorescence microscope for the transfection efficiency of cells with GFP or RNF11-GFP in each well. Relative luminescence units measured in the wells of GFP-expressing cells were then corrected for the higher expression efficiency (on average 1.5-fold higher) compared with RNF11-GFP–expressing cells.