Ha tag ip co ip kit

The lines of coexpressing hemagglutinin (HA)-labeled PIF4 (pPIF4::PIF4-HA) and YFP-labeled DCL1 (pDCL1::DCL1-YFP) were generated by crossing pDCL1::DCL1-YFP/dcl1-9 [21 (link)] with pPIF4::PIF4-HA/pif4-2 obtained by crossing pPIF4::PIF4-HA/Col with pif4-2 mutant. For control lines, plants co-expressing pPIF4::PIF4-HA/pif4-2 and p35S::YFP/Col were generated by transforming the pPIF4::PIF4-HA/pif4-2 line with the p35::YFP construct. The homozygous lines were grown in MS medium for 4 days, then seedlings were ground in liquid nitrogen and homogenized in three volumes of extraction buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 0.5% TritonX-100, 0.2% 2-mercaptoethanol, 5% glycerol) containing complete proteinase inhibitor cocktail (Roche) using a mortar and pestle set. Cell debris was pellet by centrifugation for 10 min at 13,000 g. The Co-IP experiments were performed using HA Tag IP/Co-IP Kit according to the manufacturer’s protocol (Thermo Pierce) and the previous report [21 (link)].

Plants coexpressing hemagglutinin (HA)-labeled CDF2 (pCDF2::CDF2-HA) and yellow fluorescent protein (YFP)-labeled DCL1 (pDCL1::DCL1-YFP) were generated by crossing pDCL1::DCL1-YFP/dcl1-9 [26 (link)] with pCDF2::CDF2-HA/cdf2 which was obtained by crossing pCDF2::CDF2-HA/Col with cdf2 mutant. For a control, plants co-expressing pCDF2::CDF2-HA/cdf2 and p35S::YFP were generated by transforming the pCDF2::CDF2-HA/cdf2 line with the p35::YFP construct. The homozygous lines were grown in soil for 22 days, then seedlings were ground in liquid nitrogen and homogenized in three volumes of extraction buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 0.5% TritonX-100, 0.2% 2-mercaptoethanol, 5% glycerol) containing complete proteinase inhibitor cocktail (Roche) using a mortar and pestle set [50 ]. Cell debris was pellet by centrifugation for 10 min at 15,000 rpm. The Co-IP experiments were performed using HA Tag IP/Co-IP Kit according to the manufacturer’s protocol (Thermo Pierce). Briefly, the mixed lysate was incubated with anti-HA agarose for 4 h to overnight in each spin column. The columns were washed five times with TBS plus 0.05% Tween-20 detergent (TBS-T), and resolved by SDS/PAGE. Anti-GFP (Sigma) and anti-HA (Cell signaling technology) antibodies were used to detect DCL1-YFP and CDF2-HA, respectively.