Amplify reagent

Immunoprecipitated PRMT1 from MCF-7 cells or GST-PRMT1 fusion protein were incubated with GST-hinge of ERα as described previously [4 (link)] in the presence of S-adenosyl-l [methyl-³H] methionine ([³H] SAM 85 Ci/mmol from a 10.4 mM stock solution in dilute HCl/ethanol 9/1 (pH 2.0–2.5); Perkin Elmer) for 1 h at 30 °C. Methylation reactions were quenched by adding Laemmli sample buffer, heated at 95 °C for 5 min, and separated by SDS-PAGE. Following electrophoresis, gels were soaked in Amplify reagent (Sigma) according to the manufacturer’s instructions and visualized by autoradiography. Cold experiments were performed using nonradiolabeled SAM at 0.5 mM. mERα methylation was revealed by western blotting using the anti-methyl-ERα antibody.

The PRMT5/MEP50 complex (purchased from Sigma Aldrich) was incubated with the different domains of ERα fused to GST as already described (Le Romancer et al, 2008 (link)) in the presence of S‐adenosyl‐L [methyl‐³H] methionine ([³H] SAM 85 Ci/mmol from a 10.4 mM stock solution in dilute HCl/ethanol 9/1 [pH 2.0–2.5]; Perkin Elmer) for 90 min at 30°C. Methylation reactions were quenched by adding Laemmli sample buffer, heated at 95°C for 5 min, and separated by SDS–PAGE. Following electrophoresis, gels were soaked in Amplify reagent (Sigma) according to the manufacturer's instructions and visualized by autoradiography.