Pharosfx plus

Proteins were separated using SDS-PAGE and transferred onto PVDF membranes. The antibodies were GFP ^{31 (link), 60 (link)}, FLAG (Sigma, F7425 and F3165), PiaA ^{36 (link)}, Tor (Cell Signaling, 2983), Rictor (Cell Signaling, 2114), Raptor (Cell Signaling, 2280), GAPDH (Thermo Fisher, MA5–15738), actin (Santa Cruz, Sc-1615), GSK-3 (Millipore, 05–412), phospho-RPS6KB1 (Cell Signaling, 9205), phospho-PKC (Cell Signaling, 2060), AKT (Cell Signaling, 9272) and phospho-AKT (serine 473) (Cell Signaling, 9271). Polyclonal rabbit antibodies to PKBA and PKBR1 were raised against the peptides, KNSDRKKRVNG and KKGNKNDETTP, respectively. Polyclonal rabbit antibodies to RacE and phospho-RacE (serine 192) were raised against the peptide REQQHPDPNSGKF and the phosphopeptide GMDKK(pS)QDGSS. Immunocomplexes were visualized using fluorescent-labeled secondary antibodies and detected using a Bio-Rad PharosFX Plus molecular imager. Images were analyzed using NIH ImageJ. The detailed information about the antibodies used in this study is provided in Supplementary Table 4.

WT BbRAGL or RAG proteins (25 nM final concentration of each protein), substrate DNA (final concentration 10 nM) and 175 ng His6-hHMGB1 were incubated in reaction buffer (25 mM HOPS, pH7.0, 50 mM KCl, 2 mM DTT, 1.5 mM MgCl₂; 16 μl final reaction volume) at 37°C for 1 hr or for the indicated time period. For reactions with chimeric proteins containing the RAG1 catalytic core, the final concentration of each protein was 50 nM. For reactions with chimeric proteins containing the BbRAG1L catalytic core, the final concentration of each protein was 50 nM, the Mg²⁺ concentration was 5 mM, and reaction time was 2 hr. In these experiments, control reactions for each experiment used the same conditions as the reactions with the chimeric proteins. Reactions were stopped by adding 1.25 μl 2.5% SDS, 5 μl proteinase K (150 μg/ml), 2 μl 0.5 M EDTA followed by incubation at 55°C for at least 3 hr. Samples were briefly centrifuged and the supernatant mixed with 1.7 μl 80% glycerol and loaded on a non-denaturing 1X TBE (Tris-borate-EDTA buffer) 6% polyacrylamide gel. After 1 hr electrophoresis at 100 V, gels were stained with SYBR GOLD in 1X TBE buffer for 20 min and imaged using a PharosFX™ Plus (Bio-Rad).