A4596

To examine the interaction of JANUS with DCL1 and SE, JANUS-FLAG was transiently co-expressed with GFP-DCL1 or MYC-SE in N. benthamiana as described (12 (link)). The expression of these transgenes was directed by the 35S promoter. Total proteins of infiltrated leaves were extracted with an extraction buffer (50 mM Tris–HCl 8.0, 150 mM NaCl, 5% glycerol, 5% Triton X-100, 1 mM EDTA, 1× complete protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride). Immunoprecipitation (IP) was performed on protein extracts using anti-GFP (GTA-20, Chromotek) or anti-FLAG antibodies (A4596, Sigma) coupled to protein G agarose beads. After IP, proteins were separated on a 10% SDS-PAGE and detected with western blot using monoclonal antibodies against GFP (902602, Biolegend) or FLAG (A8592, Sigma). DCL1, SE and HYL1 proteins in Arabidopsis were detected with antibodies against DCL1 (Agrisera, AS122102), SE (Agrisera, AS09532A) and HYL1 (Agrisera, AS06136).

The Co-IP assay was performed as previously described^{66 (link)}. To examine the interactions of CWC15 with HYL1, SE, PAB1 and PAG1, MYC-CWC15 was transiently co-expressed with HYL1-2HA, SE-2HA, PAB1-2HA and PAG1-2HA in N. benthamiana leaves, respectively. The interaction between CWC15 and PRP4KA was performed by co-expressing 2HA-CWC15 and FLAG-PRP4KA in N. benthamiana. IP was performed on protein extracts using anti-HA (26181, Thermo Scientific), anti-MYC (GTA020, Bulldog Bio) or anti-FLAG (A4596, Sigma) coupled to protein G agarose beads. After IP, proteins were detected with Western blot using antibodies against MYC (1:5000 dilutions, rabbit monoclonal, M4439, Sigma), FLAG (1:10000 dilutions, mouse monoclonal, A8592, Sigma) or HA (1:5000 dilutions, mouse monoclonal, H6533, Sigma). For the interaction of CWC15 and SE or HYL1 in Arabidopsis, IP was performed with ChromoTek GFP-Trap® Agarose in pCWC15::eGFP-CWC15 transgenic plant. After IP, proteins were detected with Western blot using anti-GFP (1:1000 dilutions, rabbit polyclonal, ab190584, Abcam), anti-HYL1 (1:5000 dilutions, rabbit polyclonal, AS06136, Agrisera) or anti-SE (1:5000 dilutions, rabbit polyclonal, AS09532A, Agrisera) antibodies.

Cells were incubated with 20 mM N-ethylmaleimide in PBS for 10 min on ice, snap frozen and lysed in lysis buffer (150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl (pH 7.5), protease inhibitor cocktail (Sigma–Aldrich) and 10 mM N-ethylmaleimide) supplemented with either 1% Triton X-100 or Nonidet P-40 (NP-40) on ice for 15 min. Cells were centrifuged at 12,000g at 4 °C for 10 min. Supernatants were collected and the protein concentration was measured using the Bradford assay. The protein lysates were incubated with antibodies specific for STING (19851-1AP; Proteintech) or SEL1L (ab78298; Abcam) followed by Protein A Agarose beads (20334; Invitrogen), or directly with agarose-conjugated anti-FLAG (A4596; Sigma–Aldrich), anti-Myc (16-219; Sigma–Aldrich) or streptavidin agarose (20353; Thermo Fisher Scientific) for 16 h at 4 °C with gentle rocking (1 μg antibody or 30 μl agarose beads for 1 ml sample lysis), followed by five washes with the lysis buffer. Immunocomplexes were eluted by boiling for 5 min in sodium dodecyl sulfate (SDS) sample buffer followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis.