α gfp

Ab used: α-TFIIA (sc-5314/sc-5315/sc-5311; Santa Cruz Biotechnology, Heidelberg, Germany); α-GAPDH (sc-47724; Santa Cruz Biotechnology, Heidelberg, Germany); α-GFP (sc-8334; Santa Cruz Biotechnology, Heidelberg, Germany); α-Taspase1 (sc-85945; Santa Cruz Biotechnology, Heidelberg, Germany). Appropriate HRP-, Cy3- or FITC-conjugated secondary antibodies (Sigma Aldrich, Munich, Germany; Santa Cruz Biotechnology, Heidelberg, Germany) were used. Reagents were from Sigma Aldrich (Sigma Aldrich, Munich, Germany) unless stated otherwise. Cells were treated with the export inhibitor Leptomycin B (LMB) (10 nM) as described^{54 (link)}.

Cells were washed with ice-cold PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% NP-40, 50 mM NaF, 1 mM DTT, 1 mM PMSF, and 1X Protease inhibitor cocktail). After centrifugation, the supernatant was collected for immunoprecipitation or Western blot analysis. For the Co-IP experiment, 2 mg antibody and 25 μl protein G beads were used for incubation with lysate at 4°C for 4 hrs. After 3 extensive washes with lysis buffer, the immunoprecipitated proteins were analyzed by western blot. Antibodies used include: α-β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-47778), α-GFP (Santa Cruz Biotechnology #sc-9996), α-SNRPD3 (Sigma-Aldrich #HPA001170), α-Flag-tagged HRP (Sigma-Aldrich #A8592), α-Coilin (Cell Signaling, Danvers, MA, USA, #14168), α-PARP1 (Santa Cruz Biotechnology #sd-25780), α-p53 (Santa Cruz Biotechnology #sc-126), α-phospho-p53 (pSer15) (Santa Cruz Biotechnology #sc-101762), α-p21 (Waf/Cip1, Cell Signaling #2947). The antibodies used in Co-IP were α-GFP (ProteinTech, Rosemont, IL, USA, #50430-2-AP), α-Flag (Sigma-Aldrich #M8823), α-Coilin (Abcam, Cambridge, UK, #ab87913), α-ERG (Abcam #ab92513), and α-IgG (Santa Cruz Biotechnology #sc-2025). The TDRD1 monoclonal antibody was generated in-house and published previously[1 (link)].

Probes for cyp26b1 [13 (link)] and xirp2a (cb1045, [2 (link)]) are described elsewhere. Color development for fluorescence imaging was performed with α-Digoxigenin-POD F_ab fragments (11207733910, Roche Diagnostics, Indianapolis, IN, USA) and TSA Plus Fluorescein (NEL741001KT) or Cy3 (NEL744001KT, Perkin-Elmer, Inc., Waltham, MA, USA) System. In preparation for immunohistochemistry, embryos were fixed in 95% methanol/5% glacial acetic acid. The protocol for myosin heavy chain/Tsp4b staining is described previously [11 (link)]. Primary antibodies utilized include MF 20 (1:100 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), α-Thbs4b (1:200 dilution; GTX125869, GeneTex, Inc., Irvine, CA, USA), α-GFP (1:200 dilution; sc-9996, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Alexa Fluor-conjugated secondary antibodies from Thermo Fisher Scientific (Waltham, MA, USA) were used at a 1:1000 dilution.

For transient expression, Agrobacterium cultures were grown overnight and resuspended in infiltration media (5% sucrose, 5 mM MES, 200 μm acetosyringone) to 1.0 OD₆₀₀. Arabidopsis 7-d-old AvrPto seeedlings were vacuum infiltrated (24 hr after 10 μm dexamethasone application) and collected 4 d after infiltration.
For immunoprecipitation, nuclei were first extracted with nuclei isolation buffer (0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl₂, 60 mM KCl, 15 mM NaCl, 1 mM CaCl₂, 0.9% Triton X-100, 1 mM PMSF) and resuspended in nuclei lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS, 1 mM PMSF, 1x Roche protease inhibitor cocktail) with crosslinking reagent dithiobis(succinimidyl propionate) (1 mM DSP). To quench crosslinking 50 mM Tris pH 7.5 was applied. Extracted nuclear proteins were then incubated with equilibrated GFP-trap beads (Chromotek) at 4°C for 1.5 hr under gentle agitation, followed by 3 times of washing with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl). Western blots were performed using α-GFP (Santa Cruz) or α-FLAG antibodies (Sigma).

For immunoblotting, protein samples were separated on 12% SDS-PAGE and transferred to nitrocellulose membrane (Whatman, NJ). Membranes were incubated with 7% skim milk (in PBST 1X), followed by antibody hybridization. α-Tubulin, α-Myc, α-GFP, α-HA and goat α-RIG-I antibodies were purchased from Santa Cruz Biotech. α-Mitofilin was purchased from NOVUS. α-Flag and α-V5 were purchased from Sigma and Invitrogen respectively. α-IRF3, α-RIG-I and α-FAT10 were purchased from Enzo Life Science, α-phospho IRF3 (Ser369), α-G3BP-1 was purchased from Cell Signaling. α-NP antibody was purchased from Abcam. Immunoreactive signals were detected by enhanced chemiluminescence in horseradish peroxidase (Pierce Biotechnology, MA). For immunofluorescence experiments: Alexa 488-, 568-, 647- conjugated anti-mouse, anti-rabbit, anti-goat IgG antibody purchased from Invitrogen were used as secondary antibodies.

Cell lysates were incubated overnight at 4 °C with the indicated antibodies diluted 1:200, and added to protein A/G agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA). Immune complexes were released from the beads by boiling, and then analyzed by WB. Primary antibodies used in this study: α-FLAG (F1804; Sigma-Aldrich, St. Louis, MO, USA), α-Myc (05-724MG; Millipore, Billerica, MA, USA), α-GFP (sc-9996; Santa Cruz Biotechnology), α-SIRT1 (sc-15404; Santa Cruz Biotechnology), α-PPARγ (sc-7273; Santa Cruz Biotechnology), α-β actin (sc-47778; Santa Cruz Biotechnology), α-LSD1 (ab17721; Abcam, Cambridge, UK), α-H3K9ac (ab12179; Abcam), α-H3K9me2 (ab1220; Abcam), and α-CACUL1 (rabbit polyclonal antibody raised against amino acids 338–355; Peptron, Daejeon, South Korea). The WEST-ZOL^® system (iNtRON Biotechnology, Seongnam-Si, South Korea) was used for detecting protein bands.

N. benthamiana leaves were infiltrated with A. tumefaciens AGL1 carrying binary vectors for tagged proteins. Leaf tissue was harvested at 2 dpi and snap frozen before grinding and total protein extraction. Samples were immunoprecipitated (IP) using anti-FLAG affinity gel [Sigma Aldrich, NZ]. Total protein and IP samples were boiled in loading buffer containing DTT [Sigma Aldrich, NZ] to denature proteins. SDS-polyacrylamide gel electrophoresis was performed, subsequently blotted onto PVDF membranes [Sigma Aldrich, NZ] and probed with horseradish peroxidase-conjugated antibody against the epitope tag (α-FLAG [Sigma Aldrich, NZ] or α-GFP [Santa Cruz Biotechnology, USA]) or against acetylation (α-AcK) [Cell Signaling, USA]. Visualization was achieved using Pierce Pico and Femto reagents [Thermo Fischer, NZ]. Protein loading was visualized using Ponceau staining [Thermo Fischer, NZ].