γ 32p atp

These experiments were performed as described in our previous study (59 (link), 60 (link)). Briefly, purified RavS or RavS^H503A was incubated with c-di-GMP or the purified proteins as indicated in the presence of 20 μM [γ-³²P]ATP (Perkin-Elmer, USA) in reaction buffer (50 mM Tris-HCl [pH 7.8], 25 mM NaCl, 25 mM KCl, 5 mM MgCl₂, 2 mM DTT) at 28°C for the indicated times. In RavS-RavR phosphoryl transfer assays, purified RavS or RavS^H503A were incubated with 20 μM [γ-³²P]ATP (Perkin-Elmer, USA) in reaction buffer for the indicated times at 28°C. The remaining ATP was depleted by using a PD-Spintrap G-25desalting column (GE, New York, NY) before the next step. c-di-GMP, wherever necessary, was added to the reaction system simultaneously with the other proteins. The reactions were terminated by adding 5× SDS-PAGE loading buffer. Samples were loaded into the gels for PAGE and then exposed to a phosphor screen (GE Healthcare) to detect autoradiographic signals by a Typhoon FLA7000 (GE Healthcare).

RNA gel blot analysis was carried out as previously described (Quesada et al., 2003 (link)) with minor modifications. RPP7 mRNA was detected using a probe annealing to the second exon of the RPP7 (AT1G58602) gene (200 bp PCR product amplified with the following primers: Forward: 5′-TCGGGGACTACTACTACTCAAGA-3′ and Reverse: 5′-TCTTGATGGTGTGAAAGAATCTAGT-3′). β-TUBULIN mRNA was used as a loading control and visualised by a probe annealing to the third exon of the β-TUBULIN (AT1G20010) gene (550 bp PCR product amplified with the following primers: Forward: 5′- CTGACCTCAGGAAACTCGCG-3′ and Reverse: 5′- CATCAGCAGTAGCATCTTGG-3′). The probes were 5′ labelled using [γ-³²P]-ATP (Perkin Elmer) and DECAprime II DNA labelling kit (Thermo Fisher Scientific) and purified on illustra G-50 columns (GE Healthcare Life Sciences). mRNA isoforms were visualised and quantified using an Amersham Typhoon Gel and Blot Imaging System (GE Healthcare Bio-Sciences AB). The RiboRuler High Range RNA Ladder (Thermo Fisher Scientific), used to identify the approximate size of RNA bands, was first dephosphorylated using FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) and then labelled with [γ-³²P]-ATP (Perkin Elmer) using T4 Polynucleotide Kinase (Thermo Fisher Scientific) before gel loading.

Recombinant glutathione S-transferase (GST)-MNK fusion proteins were purified by glutathione pull-down from lysates of appropriately-transfected HEK293 cells.
SGK1 protein was prepared by immunoprecipitation using anti-SGK1 antibodies. For kinase assay with [γ-³²P]ATP, beads with SGK1 protein and 300 ng non-activated MNK were incubated with 0.1 μl [γ-³²P]ATP (where used), 3000 Ci/mmol (PerkinElmer) and 0.2 μl non-radioactive ATP (10 μM) (Sigma) in kinase assay buffer (25 mM Tris-HCl pH7.5, 50 mM KCl and 2 mM MgCl₂) at 30°C for 30 min. Where indicated, HA-NDRG1 (immunoprecipitated from HEK293 cell lysates) or 250 ng eIF4E (expressed in E. coli) were used as test substrates. NDRG1 immunoprecipitates were washed twice with high salt buffer to remove contaminating kinase activity. Products were analysed by SDS-PAGE. Coomassie blue staining was used to assess protein levels and a phosphorimager scanner (PerkinElmer) to visualise the radioactive signals.
Non-radioactive assays were performed as described above, using the indicated substrates, but using only unlabelled ATP.

Total RNA (15 μg) was isolated using Trizol (Life Technologies) and resolved on a denaturing 8 M UREA/15% polyacrylamide (19:1, acrylamide:bisacrylamide) 1 × TBE gel. The gel samples were transferred to a Hybond N+ membrane (GE Healthcare) using a semi-dry apparatus (Bio-Rad) at room temperature at 300 mA for 1.5 h in 1 × TBE transfer buffer. Following transfer, the RNA was crosslinked to the membrane using a Stratalinker UV Crosslinker (Stratagene). The membrane was pre-hybridized in buffer containing 0.2 M Na₂PO₄ buffer (pH 7)/ 7% SDS for 30 min at 37 °C, at which point 40 × 10⁶ c.p.m. of radioactive probe was added and incubated overnight at 37 °C. Probes were labelled using T4 PNK (NEB) and γ-³²P-ATP (Perkin Elmer) and unincorporated γ-³²P-ATP removed by G-25 Sephadex spin columns. The following probes were used: U6 (loading control)—5′-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3′ and gRNA (targeting the scaffold portion of the gRNA) 5′-TTCAAGTTGATAACGGACTAGCCT-3′. After incubation, the membrane was washed twice with 2 × SSPE/0.1% SDS for 30 min. at 42 °C and twice with 0.5 × SSPE/ 0.1% SDS for 30 min at 42 °C. The membrane was exposed to a Phosphorimager screen for a few hours and processed using a Typhoon scanner (GE Healthcare) or exposed overnight at −80 °C to X-ray film. Quantitation was performed using ImageJ (NIH). Full uncropped images are provided in Supplementary Fig. 6.