Rabbit anti flag antibody

Whole cell lysates obtained from cells with/without AUX/DOX were subjected to western blotting. Primary antibodies used in this study are; mouse anti-C9 EWS antibody (1:1,000 dilution) (Santa Cruz, #sc-48404); rabbit anti-FLAG antibody (1:1,000 dilution) (Sigma, #F7425); rabbit anti-CyclinB antibody (1:1,000 dilution, Sigma, #C8831); mouse anti-β-actin antibody (1:1,000 dilution, Sigma, #A2228); chicken anti-mCherry antibody (1:1,000 dilution, LSBio, #C-204825). Secondary antibodies used in this study are; IRDye 680RD donkey anti-mouse IgG (1:10,000 dilution, LI-COR, #926–68072), IRDye 800CW donkey anti-rabbit IgG (1:10,000 dilution, LI-COR, #926-32213), IRDye 800CW donkey anti-mouse IgG (1:10,000 dilution, LI-COR, # 926-32212), IRDye 800CW donkey anti-chicken IgG (1:10,000 dilution, LI-COR, # 926-32218). All images of western blotting were captured by LI-COR Odyssey Imaging System.

Tissues and cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1 mM EDTA) containing a completely EDTA-free protease inhibitor cocktail (Roche), 1 mM phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded on SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Amersham Biosciences). The nitrocellulose membranes were blocked for 1 h in 5% nonfat milk in TBST (10 mM Tris, pH 7.5, 200 mM NaCl, and 0.2% Tween 20) followed by incubation with primary antibodies. Mouse anti-GFP antibody (Clontech), rabbit anti-GFP antibody (Clontech), mouse anti-FLAG antibody (Sigma), rabbit anti-FLAG antibody (Sigma) and mouse anti-CaMKIV (Abnova) were used, and the bound antibodies were visualized by Lumi-Phos WB Chemiluminescent Substrate (Thermo Scientific).

Total proteins were harvested from leaf samples as described previously by Sun et al. (2018) using 2× SDS loading buffer (100 mm Tris (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue) containing 10% β-mercaptoethanol. Proteins were isolated on 12.5% SDS polyacrylamide gels and then transferred onto polyvinylidene fluoride membranes (Ge Healthcare, Buckinghamshire, UK). The membranes were blotted with a rabbit anti-flag antibody (1:1000; Sigma–Aldrich, St. Louis, MO, USA), mouse anti-flag antibody (1:5000; Sigma–Aldrich), rabbit anti-GFP antibody (1:3000; Genscript, Nanjing, China) or rabbit anti-PLRV MP antibody (1:5000) [53 (link)]. Immunoreactive proteins were successively visualized by blotting with goat anti-rabbit AP antibody (1:10,000; Sigma–Aldrich), goat anti-mouse AP antibody (1:10,000; Sigma–Aldrich), goat anti-rabbit HRP antibody (1:3000; Sigma–Aldrich) or goat anti-mouse HRP antibody (1:3000; Bio-Rad, Hercules, CA, USA) followed by visualization using nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate (Sigma–Aldrich) or a chemiluminescence detection kit (GE Healthcare).

HEK 293T cells in a 10-cm Petri dish were transfected with myc-FTL, flag-Gadd45a, and their control expression vectors in different combinations using the Attractene Transfection Reagent (Qiagen). Forty-eight hours after transfection, cells were rinsed twice with PBS and lysed in 1 ml lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.5% Nonidet P-40 with proteinase and phosphatase inhibitors. The cell lysates were briefly centrifuged to remove cell debris, and the supernatant was separated into three aliquots (300 μl per aliquot). The first aliquot was incubated with 10 μl of mouse anti-myc monoclonal antibody (Santa Cruz Biotechnology: sc-40), the second aliquot was incubated with 2 μl of mouse anti-flag monoclonal antibody (MBL, Nagoya, Japan: M185-3L), and the third aliquot was incubated with 2 μl of mouse monoclonal anti-IgG antibody (Abcam: Ab18413). After incubation at 4°C overnight, the immune complex was precipitated with 20 μl Protein G Plus-Agarose (Santa Cruz Biotechnology: sc-2002). The immunoprecipitates were washed four times with lysis buffer, and then subjected to electrophoresis. The antibodies used for western blotting were rabbit anti-myc antibody (Santa Cruz Biotechnology: sc-789) and rabbit anti-flag antibody (Sigma-Aldrich: F7425).