Sc 8036

Mtb cells were lysed with TRIzol reagent as described above, and protein samples were extracted following a TRIzol-based protein extraction protocol provided by the manufacturer. Immunoblotting was carried out as described previously^{48 (link)}. Antibodies against His-tag (Santa Cruz, sc-8036; 1:1,000 dilution), Mtb Rho (obtained from D. Schnappinger; 1:200 dilution), and Eco RpoB (BioLegend, 663903; 1:1,000 dilution) were used.

Prp16p (3 μg) and Sec63-2p (1.5 μg) were incubated overnight at 4°C with protein A dynabeads (Roche) and anti-Prp16p-029 (Eurogentec, this study) at 1/100 dilution in IP buffer (50-mM Tris–HCl, pH 7.5, 150-mM NaCl, 1-mM MgSO₄, 0.02% (v/v) NP-40) containing bovine serum albumin (BSA) (100 μg/ml), glycogen (50 μg/ml) and tRNA (100 μg/ml). For co-immunoprecipitations treated with RNase A (30 ng/μl), tRNA was omitted. After extensive washing with IP buffer, beads were loaded onto NuPAGE 4–12% gradient gels (Invitrogen), followed by western blotting using anti-His-HRP-conjugated antibodies (sc-8036, Santa Cruz; 1/3000 dilution).

To examine the interactions between various PRPK mutants with TPRKB or OSGEP, GST-tagged PRPK proteins were first captured onto 20 µl glutathione Sepharose 4B resin from an appropriate amount of BL21 lysates. Then, beads with bound GST-PRPK proteins were incubated with approximately 15 µg purified His-TPRKB or MBP-OSGEP proteins in binding buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% Nonidet P-40, 5% glycerol, 2 mM DTT, and 0.4 mM phenylmethanesulfonyl fluoride (PMSF)) for 2 h at 4°C. Beads were washed with binding buffer 4 times, and the bound proteins were detected by Western blotting by using anti-His (Santa Cruz, sc-8036; Santa Cruz, CA, USA) or anti-MBP (Cell Signaling #2396; Danvers, MA, USA).
To test the binding between MTX and PRPK, Methotrexate-agarose suspension from Sigma (Catalogue number M0269) was used. Glutathione-agarose resin was used as control beads. Purified PRPK–TPRKB protein (10 µg) was incubated with 20 µl compound-beads (or control beads) in binding buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% Nonidet P-40, 5% glycerol, 2 mM DTT, and 0.4 mM PMSF) for 2 h at 4°C. Beads were washed with binding buffer 4 times, and the bound proteins were detected by Western blotting by using anti-PRPK (Santa Cruz, sc-514703).

For western blot analysis, proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (0.45 μm; Bio-Rad Laboratories). Membranes were blocked in 5% non-fat dry milk diluted in Tris-buffered saline with 0.1% Tween (TBS-T) buffer. The following primary antibodies, diluted in TBS-T buffer, were used: anti-eIF6 (1:1000, overnight, D16E9, Cell Signaling), anti–His (1:1000, overnight, sc-8036, Santa Cruz Biotechnology). Anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (1:30 000, 1 h, Jackson Immunoresearch) were also used. For Ponceau S staining, membranes were rinsed in ultrapure water and stained with Ponceau S dye and de-stained with a brief rinse in ultrapure water. Gels and blots were imaged using iBright FL1500 imaging system (Fisher Scientific).