P nitrobenzyl mesylate

Purified proteins were incubated with kinase buffer containing 20 mM Tris–HCl pH 7.5, 10 mM MgCl₂, 1× protease inhibitor cocktail (Roche), 1 μM okadaic acid, 1 mM DTT, 100 μM ATP and 0.5 mM ATPγS or 6‐benzyl‐ATPγS (Biolog) at 30°C. Analog‐sensitive CDKL5 auto‐thiophosphorylation was determined with ~250 ng (200 nM) CDKL5^1–352 on HA‐beads for 30 min. All substrate thiophosphorylation was done with insect cell‐purified AS‐CDKL5, 6‐benzyl‐ATPγS and 150 ng (50 nM) MAP1S, 200 ng (170 nM) EB2, 150 ng (50 nM) ARHGEF2 or 300 ng (130 nM) AMPH1. Time courses were performed with 50 ng (40 nM) AS‐CDKL5 for 0–60 min. Titrations with 0–50 ng AS‐CDKL5 incubated for 30 min (MAP1S, EB2) or 60 min (AMPH1, ARHGEF2). Phosphomutant experiments incubated as long as the titrations and contained analog‐sensitive and kinase‐dead CDKL5^1–352 at the following concentrations: 50 ng (AMPH1), 5 ng (MAP1S), 10 ng (EB2), 20 ng (ARHGEF2). Reactions were quenched with 20 mM EDTA and followed with alkylation by 5 mM p‐nitrobenzyl mesylate (Abcam) for 1 h at RT. Proteins were solubilized by sample buffer containing 0.1 M DTT. Thiophosphorylation was detected by anti‐thiophosphate ester antibody on Western blots.

HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, cells were lysed in 1.0 mL IP lysis buffer supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 17,700 × g for 10 min at 4°C. The supernatant was incubated with 50 μL anti-HA magnetic beads for 2 h at 4°C. The beads were washed five times with IP lysis buffer and once with kinase buffer (40 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, and 50 μM dithiothreitol [DTT]). The immunocomplex was incubated with GST-IκBα (catalog no. ab59981; Abcam) as the substrates in kinase buffer with 1 mM ATP-γ-S (catalog no. ab138911; Abcam) at 37°C for 30 min. Reactions were followed by addition of 2.5 mM p-nitrobenzyl mesylate (catalog no. ab138910; Abcam) to start alkylation for 2 h at room temperature. The reaction was stopped by boiling in 4× sample buffer and subjected to Western blotting. Membranes were probed with anti-thiophosphate ester antibody and horseradish peroxidase-conjugated secondary antibody. Labeled proteins were visualized using a LuminoGraph I.

For Western blotting, anti-α-tubulin (T6199, 1:5000 dilution) and anti-Flag (M2) (F3165-5MG, 1:5000 dilution) antibodies were obtained from Sigma-Aldrich. Anti-Myc (sc-40, 1:500 dilution) and anti-GST antibodies (sc-138, 1:1000 dilution) were purchased from Santa Cruz Biotechnology. Anti-phospho-YAP (S127) (4911S, 1:1000 dilution) and anti-phospho-LATS1 (Thr1079) (8654S, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology. Anti-hemagglutinin (HA) monoclonal antibody (901514, 1:2000 dilution) was obtained from BioLegend. Anti-Thiophosphate ester antibody (ab92670, 1:1000 dilution) was purchased from Abcam. The YAP and MBP polyclonal antibodies were generated as previously described^{57 (link),61 (link)}. ATP-γ-S kinase substrate (ab138911) and p-Nitrobenzyl mesylate (ab138910) were obtained from Abcam. For immunostaining, an anti-YAP (sc-101199, 1:200 dilution) monoclonal antibody was purchased from Santa Cruz Biotechnology. Anti-Flag polyclonal antibody (F7425, 1:5000 dilution) was obtained from Sigma-Aldrich.