Anti actin

Total proteins were extracted using extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 0.5 mM PMSF, 10% glycerol, 10 mM DTT and protease inhibitor cocktail). The protein samples were separated by 12% SDS-PAGE gels. After separation by electrophoresis, proteins were transferred onto the PVDF membrane and detected using the primary antibodies anti-H3K9K14ac (Millipore, Cat No. 06-599), anti-H3K4me³ (AbCam Cat No. ab8580), and anti-Actin (Abbkine Cat No. A01050). Quantification of the band intensities on the western blot was performed using the Image J software.

After PEG-mediated transformation, the rice protoplasts were placed in 1 ml W5 buffer containing 10 μM 3-MA (3-methyladenine, Selleck, S2767), 100 μM chloroquine (MCE, HY-17589A), 100 μM leupeptin (MCE, HY-18234A), or 20 μM E-64d (Aloxistatin, Sigma-Aldrich, E8640) to inhibit autophagy or 50 μM MG132 (Selleck, S2619) to inhibit the 26S proteasome. The treated protoplasts were incubated at 28 °C for 14–20 h before protein extraction. Anti-actin (Abbkine, ABL1050) was used as a reference to quantify total protein levels. Rice WRKY72 is degraded by the 26S proteasome pathway^{19 (link)} and was used as a positive control to ensure that the proteasome inhibitor MG132 was active. The rice HD1 protein is degraded in the dark by the autophagy pathway^{61 (link)}, and OsNBR1 was used as a positive control to assure autophagy inhibitors were active. The intensity of protein signals in immunoblots was quantified by ImageJ.

Tea samples with or without tea green leafhopper infestation were harvested for total protein extraction. The sample powder was fully homogenized with vegetable protein extraction buffer (Fude, China), then boiled and separated on Smart PAGE™ precast gels using MOPS (3-(N-morpholino)propanesulfonic acid) running buffer (Smart-Lifescience, China). Anti-Actin (Abbkine, USA) and anti-CsHDA2 (Huaan, China) antibodies were used as primary antibodies, and appropriate goat secondary antibodies conjugated to HRP (horseradish peroxidase) (Abbkine, USA) were used for detection. The gray-scale intensity of protein bands in western blots was analyzed by Image Lab software (Bio-Rad, USA).