Amplify solution

Uncapped Top7 mRNAs were translated in nuclease-treated RRL (Promega). The 500 ng mRNA was incubated in 8.4 μL RRL, 0.24 μL 1 mM amino acid mixture minus methionine, 0.48 μL ³⁵S methionine, 0.24 μL RNasin, and ddH₂O to 20 μL. Translation reactions were incubated at 30°C for 90 minutes and quenched with 20 μL 2X SDS sample buffer. Translation reactions were then incubated at 60°C for 20 minutes and resolved on a 14% to 20% SDS-PAGE gel. Gels were fixed in 30% methanol, 10% acetic acid, incubated in Amplify solution (GE Healthcare, Marlborough, MA), and dried on a vacuum drier. Dried gels were exposed for a minimum of 12 hours on a storage phosphor screen (GE Healthcare), scanned (Bio-Rad), and quantified using GelAnalyzer 19.1 (www.gelanalyzer.com).

After SDS-PAGE separation of methylation assay components, gels were soaked in Amplify solution (GE Healthcare, Chicago, IL) for 30 min before drying on a vacuum air dryer. Dried gels were exposed to X-ray film for 1 week at –80°C. Images were developed and fixed using Kodak (Rochester, NY) GBX solutions.

Recombinant methylosome consisting of baculovirus-expressed PRMT5/MEP50 and bacterially expressed pICln (1 pmol each) was mixed in 20 µL of the cytoplasmic buffer with a 2- to 10-fold excess of recombinant Sm proteins, and each sample was supplemented with 2 µCi of ³H-labeled SAM (PerkinElmer) for overnight methylation at 32°C. Each sample was mixed with an equal volume of 2× SDS sample buffer, resolved in 15% or 4%–12% SDS/polyacrylamide gels. Separated proteins were visualized by Coomassie Blue staining and gel images captured. The stained gels were incubated for 30 min with Amplify solution (GE Healthcare), dried and used for 12- to 72-h fluorography at −80°C. Methylated proteins were identified by aligning fluorograms with stained gel images.

Purified cell walls, previously labeled with [¹⁴C]lysine, were sequentially extracted with SDS, zymolyase (Miles Laboratories), and chitinase (Sigma) as described (24 (link), 54 (link)). Briefly, the treatment with 2% SDS was performed in a boiling water bath for 10 min, and solubilized proteins were recovered by centrifugation at 3000 × g for 10 min at room temperature. The remaining insoluble material was washed by centrifugation with water, ethanol, and water, and this process was repeated. The material was treated with 1 mg/ml zymolyase 20T containing 1 mm PMSF at 30 °C for 3 h, and solubilized material was separated by centrifugation. Finally, the pellet was treated with 0.5 mg/ml chitinase in 10 mm phosphate buffer, pH 7.0, at 30 °C for 3 h, and solubilized material was collected. Radioactivity in all samples was quantified as above. Samples extracted with SDS were precipitated with 75% ethanol at 4 °C and washed three times with ethanol by centrifugation. Samples containing 10,000 cpm were electrophoresed in 10% SDS-polyacrylamide gels, stained with Coomassie Brilliant Blue, incubated with Amplify solution (GE Healthcare) as recommended, dried, and exposed to Kodak X-Omat S films at −70 °C for 1 month (24 (link)).