Chromotek gfp trap

Co-IP was performed as described previously with few modifications^{32 (link)}. In brief, ~3 g leaf tissues were ground into fine powder in liquid nitrogen, mixed with 15 mL IP buffer I (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 0.1% NP-40, 5 mM EDTA, 2 mM dl-dithiothreitol [DTT], 5% glycerin, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1/2 tablet of Complete Protease Inhibitor [Roche, Shanghai, China]), and placed on ice for 15 min. After being filtered through two layers of Miracloth (Merch Millipore), the solution was sonicated 10 s at 10% power level eight times with a 1 min interval. The lysate was then centrifuged at 6000×g for 25 min at 4 °C. The resulting supernatant was added to 30 μL of Chromotek GFP-Trap (Proteintech, Wuhan, Hubei, China) or anti-FLAG M2 affinity gel (Sigma‒Aldrich, Shanghai, China) and incubated for 3 h at 4 °C with gentle rotation. Agarose beads were collected by centrifugation, washed at least five times with IP Buffer and resuspended in 50 µL 1× SDS‒PAGE loading buffer. The solution was frozen at −80 °C until use or directly used for SDS-PAGE.

4′,6-Diamidino-2-phenyl-indole (DAPI) (C0065) was purchased from Solarbio (Beijing, China). ChromoTek GFP-Trap (gta) and DYKDDDDK Fab-Trap (ffa) were purchased from Proteintech (Wuhan, China). BeaverBeads GSH (70601-K10) and His tag (70501-K10) were purchased from Beaverbio (Suzhou, China). TRIzol reagent (15596018), Lipofectamine 2000 (11668019), and Lipofectamine RNAiMAX (13778150) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). A2ti-1 (HY-136465) and PP2 (HY-13805) were purchased from MedChemExpress (South Brunswick, NJ, USA).