Anti myc hrp

Agrobacterium tumefaciens GV3101 carrying the indicated constructs in infiltration buffer (10 mM MES pH 5.7, 10 mM MgCl₂, 150 μM acetosyringone) was infiltrated into 3-week-old leaves of N. benthamiana. After 2 days, 200 μM of ATP and 2 mM MES (pH 5.7) were infiltrated into the same leaves. Total protein was extracted from pulverized (ground in liquid nitrogen) N. benthamiana leaf tissues using the following buffer: 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 10% glycerol, 0.5% Triton-X 100, and 1X protease inhibitor (Thermo Fisher Scientific) by gentle rotation at 4 °C for 2 h. The solution was centrifuged at 20,000 g for 10 min at 4 °C. The supernatant was transferred into a new tube and 1 μg anti-Myc-HRP (Santa Cruz, Cat.No.sc-40) was added, and incubated overnight with end-to-end shaking at 4 °C. Subsequently, 25 μl protein A resin (GenScript) was added for 4 h, spun down and washed five times with washing buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1X protease inhibitor. After washing, the resin was eluted with 50 μl 1X SDS-PAGE loading buffer and the eluent heated in boiling water for 10 min. The proteins were separated by 10% SDS-PAGE gel electrophoresis and detected by immunoblotting with both anti-HA-HRP (Roche, Cat. No.12 013 819 001, dilution 1:1000) and anti-Myc-HRP (Santa Cruz, Cat.No.sc-40, dilution 1:1000).

Western blot analysis was used to confirm the presence of proteins in N. benthamiana during cell death assays. Three leaf discs were taken at 2 dpi, flash-frozen in liquid nitrogen, and ground to a fine powder using a micropestle. Leaf powder was mixed with 300 μl of plant protein extraction buffer (GTEN (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 % v/v glycerol), 10 mM DTT, 2% (w/v) PVPP, 0.1% Tween–20, 1 x plant protease inhibitor cocktail (Sigma)). The sample was clarified by centrifugation (20,000 × g for 2 min at 4 °C, twice) and 40 μL of the resulting supernatant was added to 10 μL SDS-PAGE loading dye (RunBlue 4 x LDS sample buffer (Expedeon)), followed by incubation at 95 °C for 5 min.
Samples were subjected to SDS-PAGE/western blot analysis to detect epitope-tagged proteins. Pikp-1, Pikp-2, and AVR-Pik effectors were detected by probing membranes with anti-FLAG-HRP (Generon, 1:10,000 dilution), anti-HA-HRP (Thermo Fisher Scientific, 1:3000 dilution) and anti-Myc-HRP (Santa Cruz, 1:5000 dilution) antibodies, respectively, and LumiBlue ECL Extreme (Expedeon). Membranes were also stained with Ponceau S to observe protein loading.

Cells were lysed using RIPA buffer (Tris-HCl pH 7.6 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, sodium pyrophosphate 2 mM and protease inhibitors). Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures. The following antibodies were used: anti-Arrb1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-Nanog (Cosmo Bio Co, Japan), anti-Actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-β-III-Tubulin (MAB 1637 Millipore), anti-Gli1 H-300 (sc-20,687; Santa Cruz Biotechnology), anti-acetyl-Gli1 (Lys518) (Eurogentec) [32 (link)], anti-p300 C-20 (sc-585; Santa Cruz Biotechnology), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma), anti-HA (sc-7392 Santa Cruz), anti-Gli2 H-300 (sc-28,674; Santa Cruz Biotechnology), anti-Smo N-19 (sc-6366; Santa Cruz Biotechnology), anti-Sox2 (MAB4343 Millipore). HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used in combination with enhanced chemo-luminescence (ECL Amersham).
For immunoprecipitation assay antibody sources and concentrations used were: Protein G Plus-Agarose (sc-2002; Santa Cruz Biotechnology); anti-FLAG M2 Affinity Gel (Sigma A2220, IP 30⌠l), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma, western blotting 1:5000), anti-HA (sc-7392 Santa Cruz, 1:1000); anti-myc-HRP.