Bovine serum albumin (bsa)

DNA was extracted from dried specimens using a commercial DNA extraction kit (E.Z.N.A. Forensic Kit, D3591-01, Omega BioTek). The polymerase chain reaction (PCR) reactions and sequencing were performed using the primers ITS5 and ITS4 (White et al. 1990). The PCR reaction contained 5 μL PCR GoTaq buffer (5X, Promega), 2.5 μL deoxy-ribonucleotide triphosphate mix (1.2 mmol/L, Eurobio), 0.5 μL bovine serum albumin (10 mg/mL, Promega), 1 μL of each primer (25 mmol/L), 0.2 μL Taq polymerase (5 U/μL, GoTaq Promega), 1 μL DNA template, and ddH₂O up to 25 μL. PCR thermal cycling conditions followed those of Zhao et al. (2011) with some modifications. The programme was 5 min at 95°C; 35 cycles (denaturation 1 min at 94°C, annealing 1.5 min at 52°C, extension 1.5 min at 72°C); 5 min at 72°C for the final extension. The PCR products were examined electrophoretically in an agarose gel stained with ethidium bromide (EB), then sequenced by Biomed Co. Ltd., using an ABI 3730XL Analyzer and ABI BigDye 3.1 Cycle Sequencing Kit.

The PPP2CA catalytic subunit or the PP2A complex encompassing PPP2CA, PPP2R2A, and PPP2R1A was immunopurified from 293T cells transfected with pFlag-CMV2-PPP2R2A, pFlag-CMV2-PPP2R1A, and pcDNA3-HA-PPP2CA. Overall, 0.5 M NaCl was used in the washing buffer to remove PP2A-associated proteins. Flag-BRAF proteins were purified as described above in the In vitro kinase assays section. PP2A activity was assayed by incubating the immunopurified PP2A proteins with Flag-BRAF in the phosphatase assay buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 0.25 mg∙ml⁻¹ bovine serum albumin) at 30 °C for 60 min prior to detection with malachite green phosphate detection solution (Promega)^{55 (link)}.

SLIVm RNA was biotinylated using the Pierce RNA 3′ End Biotinylation Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA mobility shift assays were performed as described previously (35 (link),82 (link)). Purified PCBP2-FL or PCBP2-ΔKH3 were preincubated with 1 mg/ml tRNA (Roche) in binding buffer (5 mM HEPES-KOH pH 7.5, 25 mM KCl, 2.5 mM MgCl₂, 3.8% glycerol, 20 mM DTT) for 10 min at 30°C. Biotinylated RNA probe and 8 U of RNAsin (Promega) were then added followed by incubation for 10 min at 30°C. The reaction mixture was further incubated with 0.5 mg/ml of bovine serum albumin (Promega) for 10 min at 30°C. Following incubation, 1.25 μl of 80% glycerol was added. The samples were separated by native TBE-PAGE containing 5% acrylamide at 4°C, electrophoretically transferred to a nylon membrane (Roche), UV-crosslinked to the membrane using a Stratalinker UV Crosslinker 1800 (Stratagene, USA). The biotinylated RNA on the membranes was visualized using a LightShift Chemiluminescent RNA EMSA kit (Thermo Fisher Scientific) on a Chemidoc Touch Imaging System (Bio-Rad) and quantified using ImageQuant TL 8.1 software (GE Healthcare). The fraction bound was calculated as the intensity of bound SLIVm RNA as a fraction of the total intensity of bound and free SLIVm RNA probe.