Monoclonal anti flag m2 antibody produced

Mouse monoclonal anti-ICP8 (sc-53329), mouse monoclonal anti-PDCD4 (sc-376430), and rabbit polyclonal anti-H3 (sc-10809) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-eIF2α (9722), rabbit monoclonal anti-phospho-eIF2α (Ser 51) (3398), rabbit polyclonal anti-phospho-eIF4E (Ser 209) (9741), rabbit polyclonal anti-PKR (3072), and rabbit polyclonal anti-phospho-PKR (Thr 446) (3076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal against NOP53 (ab131002) was purchased from Abcam (Cambridge, MA, USA). mAb 12D10 against puromycin was purchased from Merck Millipore. Rabbit polyclonal to eIF4E (11149-1-AP) was purchased from proteintech (Wuhan, China). Monoclonal ANTI-FLAG® M2 antibody produced in mouse (F1804) was purchased from Sigma-Aldrich. Mouse monoclonal anti-Actin (AA128), mouse monoclonal anti-GFP (AG281), goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) (A0216), goat anti-rabbit secondary antibodies conjugated to HRP (A0208) used for Western blotting were all purchased from Beyotime Biotechnology. Goat anti-rabbit IgG (H + L) secondary antibody conjugated to Alexa Flour 594 (R37117) and goat anti-mouse IgG (H + L) secondary antibody conjugated to Alexa Flour 488 (R37120) used for immunofluorescence were purchased from Invitrogen.

To test physical association between BSL2 or BSU1 with MPK6 in plant cells, total cell proteins were extracted from N. benthamiana leaves that transiently expressed the combinations of 35Sp::YFP or 35Sp::MPK6-YFP with 35Sp::BSL2-FLAG or 35Sp::BSU1-FLAG. N. benthamiana leaves were collected and ground in liquid nitrogen with the protein extraction buffer (100 mM Tris-HCl pH 7.5, 5 mM EDTA, 5 mM EGTA, 1 mM Na₃VO₄, 10 mM NaF, 50 mM b-glycerophosphate, 10 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 5% (v/v) glycerol, 0.5% (v/v) Triton X-100, and 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich, P 9599)). Protein extracts were centrifuged at 18,000 × g at 4 °C for 30 min and the supernatants were incubated with GFP-Trap Agarose beads (Chromotek) at 4 °C for 3 h. Then, the beads were washed three times with extraction buffer, followed by mixing with 2 × SDS sample buffer and boiling for 5 min. Samples were separated by 10% SDS–PAGE and analyzed by corresponding primary antibodies (Monoclonal ANTI-FLAG M2 antibody produced in mouse, F3165, Sigma-Aldrich; anti-GFP Antibody, Roche #11814460001).