Ab92570

HA-tagged CDH1 WT and mutants were transfected into HEK293T cells, followed by being immunoprecipitated with monoclonal Anti-HA-Agarose antibody (Sigma, A2095). The purified HA-CDH1 proteins were then incubated with 500 uM of ATPγS (Abcam, ab138911) and 0.5 ug of recombinant human cyclin D1 + CDK4 proteins (Abcam, ab55695) in the kinase reaction buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35, pH 7.5) for 30 min at room temperature. Then adding 2 mM of PNBM (Abcam, ab138910) and allowing the alkylating reaction proceed for additional 2 h at room temperature. The reaction was then terminated by adding 5x SDS loading buffer and boiled for 10 min. Samples were then subjected to IB using anti-Thiophosphate ester antibody (Abcam, ab92570).

Phosphorylation of WalK was analyzed using an ATPγS-based protocol (Carlson et al., 2010 (link)). WalK proteins at 5 μM were incubated with 100 μM ATPγS in a kinase reaction buffer (20 mM Tris–HCl, pH 7.4, 100 mM NaCl, 50 mM KCl, 2 mM MgCl₂, and 20% v/v glycerol) for 30 min at 37°C, and terminated by 20 mM EDTA. The mixtures were added with 2.5 mM para-nitrobenzylmesylate (PNBM) (ab138910, Abcam, Cambridge, United Kingdom) to alkylate ATPγS for 1 h, at RT. The resulting products were separated by 12% w/v SDS-PAGE and transferred to PVDF membrane at a constant voltage of 100 V for 60 min at 4°C using transfer buffer (25 mM Tris-HCl, pH 8.0,192 mM glycine, and 10% v/v methanol), which was blocked with 5% w/v milk in TBST [20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% (v/v) Tween 20] for 1 h, and incubated overnight at 4°C with the first antibody (1:5,000) (Rabbit monoclonal anti-Thiophosphate ester antibody, ab92570, Abcam, Cambridge, United Kingdom). The blot was washed with TBST and incubated for 1 h at RT with the secondary antibody (1:2,500) (Goat anti-rabbit IgG-peroxidase antibody, A154, Proteintech, China). Protein targets were finally developed using chemiluminescence (WesternBright ECL, K12045, Advansta, San Jose, CA, United States) and imaged in Gel Doc XR + System (Bio-Rad, United States).

In vitro phosphorylation assays were performed as previously described [57 (link)]. Recombinant glutathione S-transferase (GST)-OsMKK6 or GST-OsMKK6^DD (500 ng) was incubated with the substrate proteins maltose binding protein (MBP)-OsMPK4 or MBP-OsMPK4m (1 μg) in kinase reaction buffer (30 mM Tris-HCl pH 7.5, 1 mM EGTA, 10 mM MgCl₂, 1 mM DTT, and 3 μL 10 mM ATPγS [Abcam, ab138911]) at 30 °C for 30 min. After adding 1.5 μL 50 mM p-Nitrobenzyl mesylate (PNBM, Abcam, Cambridge, UK; ab138910) to the reactions, the samples were incubated at 30 °C for 1.5 h. Recombinant proteins were separated by 10% (w/v) SDS-PAGE, and phosphorylated recombinant MKK6, MKK6^DD, MPK4, and MPK4m were detected by immunoblotting with anti-thiophosphate ester rabbit monoclonal antibodies (Abcam, Cambridge, UK; ab92570, 1:5000).
To detect MAPK activation, WT and rsr25 plants were inoculated with Xoo, and total proteins were extracted from the plants at 0 h, 48 h, 96 h, and 120 h after inoculation. MPK activation was detected by immunoblotting with an anti-p44/42 ERK antibody (Cell Signaling, Danvers, MA, USA; 4370S).

Samples were boiled for 5 min at 95 °C and subjected to SDS-PAGE. Immunoblotting was performed using primary antibodies diluted in phosphate buffered saline containing 0.1% Tween and 5% nonfat dry milk. myc-tagged ALPK1, GST-TIFA and thiophosphorylated proteins were detected with a mouse monoclonal anti-myc antibody (9E10, Santa Cruz Biotechnology), a rabbit polyclonal anti-GST antibody (Abcam, ab19256) and a rabbit polyclonal anti-thiophosphate ester antibody (Abcam, ab92570), respectively. HRP-conjugated secondary antibodies were purchased from GE Healthcare, Cell Signaling technologies or ThermoFisher Scientific. Blots were analysed with an enhanced chemiluminescence method (SuperSignal West Pico Chemiluminescent substrate, Thermo Fisher Scientific). As indicated in the figure legends of the Supplemental information section, some membranes were stripped in stripping buffer (Thermo Fisher Scientific) for 30 min at 37 °C and immunoblotted again as described above.

Recombinant proteins of Flag-ARID1A (substrate) and HA-IKKβ (enzyme) were first prepared and purified from E. coli. In a typical phosphorylation reaction, 1 μg Flag-ARID1A protein was incubated with HA-IKKβ in a 50 μl kinase reaction buffer (50 mM Tris-HCl, 5 mM MgCl2, 30 μM ATP) at 37 °C for 1 h. Phosphorylation of ARID1A was analyzed by western blotting with a thiophosphate ester rabbit monoclonal antibody (Abcam; ab92570). Full scan blots, see the Source Data file.