Anti ha hrp

Western blot analysis was performed as described [5 (link), 18 (link)]. The primary antibodies used for Western blot analysis were anti-phospho-serine (Chemicon), β-actin (Santa Cruz), anti-Flag-HRP (Sigma), anti-HA-HRP (Sigma), anti-myc-HRP (Sigma), anti-SPDEF (Santa Cruz), and anti-CDK11p58 (Santa Cruz).

HEK293 cells were grown in RPMI supplemented with 10% FBS at 37°C in 5% CO₂. HEK293 cells were transfected with HA–TEAD2 and/or Myc–VGLL3, as indicated. The tags were introduced to the 5′ end through modification of the pCI-neo vector (Promega). Constructs were transfected using Lipofectamine 2000. At 24 h post transfection, the cells were lysed in buffer with 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1% Triton X-100 and protease inhibitors. HA–TEAD2 was immunoprecipitated using anti-HA beads (Sigma) and the co-immunoprecipitated YAP was detected using anti-YAP antibody (D8H1X, Cell Signaling). HA–TEAD2 was detected using anti-HA-HRP (Sigma) and Myc–VGLL3 was detected using clone 4A6 antibody (Millipore). For full details of antibodies, see Table S8.
Labelled human YAP (amino acids 61–100) and unlabelled human VGLL3 (amino acids 86–112) peptides were used. Histidine-tagged human TEAD4 (amino acids 217–434) was expressed in E.coli and purified by using immobilized metal affinity chromatography (IMAC) and gel filtration chromatography. TEAD4 was incubated with an increasing concentration of VGLL3 peptide prior to the addition of labelled YAP. After a 10-min incubation at room temperature, a native gel (10%) was run at 100 V to visualize free and TEAD-bound YAP.

Spleen cells from a C57BL/6 mouse were incubated at 37°C for 1 h with or without 10 μM HA-LPETG and with or without 1 μM 7+ or 7M SrtA. The reaction mixture was then boiled in SDS loading buffer at 95°C for 10 min, run on a 12% Tris-glycine polyacrylamide gel, and transferred to PVDF. The blot was probed using anti-HA-HRP (Sigma) at a 1:2000 dilution. As a loading control, the blot was stripped and re-incubated with an anti-beta tubulin-HRP antibody (CST) at 1:1000.

Transiently transformed leaf tissue from Nicotiana benthamiana was infiltrated with 1 µM flg22 or 1 µM elf18, or with water for mock infiltration. After 2 minutes the leaf tissues were blotted dry and frozen in liquid N₂. Then 200 mg of tissues were ground in 200 µl protein extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1x plant protease inhibitor cocktail (Sigma-Aldrich)). After centrifugation 300 µl supernatant was incubated with 3 µl 9E10 anti-myc antibody (Sigma-Aldrich or Covance) and rotated at 4°C for 1 h. 50 µl Protein A (Thermo Scientific) was added and the tubes were rotated at 4°C for an additional 2 h. After 3x washing with protein extraction buffer and 1x washing with ddH₂O the beads were resuspended in 60 µl loading buffer and boiled at 95°C for 5 min. After centrifugation the supernatant was separated on two 8% SDS-PAGE gels. For protein detection the antibodies anti-HA-HRP, anti-myc rabbit and goat-anti-rabbit-HRP (Sigma-Aldrich) were used.

Hygromycin (merckmillipore), anti-HA HRP (sigma), ^3HTaurocholate (Perkin Elmer), Taurocholate (Sigma), MG132 (Z-Leu-Leu-Leu-al) (sigma), Bafilomycin A1 (sigma), DMSO (VWR), Myrcludex B-FITC (Pepscan), Transferrin receptor (Invitrogen), anti-mouse-HRP(DAKO).

The effector genes cloned in pEG101‐SacB/R could express the effecor‐YFP‐HA fusion proteins, which can be detected with anti‐HA antibodies by Western blot. At 20 h after Agrobacterium infiltration, the transiently expressed effecor fusion proteins were extracted by grinding three leaf discs (19 cm diameter) in 100 µL 3 × Laemmli buffer containing 16% β‐mercaptoethanol. Also, 20 µL protein samples were separated on a 10% SDS‐PAGE gel, and blotted to a PVDF membrane. The blot was probed with anti‐HA‐HRP (Sigma, St. Louis, MO; H6533; 1:500), and the signal was detected with using SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). The chemiluminescent signals were exposed to autoradiography film (Genesee Scientific, San Diego, CA) using a Kodak film processor (Kodak, A Walsh Imaging, Inc, Pompton Lakes, NJ). Another 20 µL protein samples were also separated on a 10% SDS‐PAGE gel, and stained with comassie blue to confirm the equal loadings.