Ha probe f 7 hrp

B16F1 and 3LL cells (4 × 10⁷ cells) treated with or without N-glycosidase (25 U/ml) and B16F10 cells and MEFs (4 × 10⁷ cells) treated without N-glycosidase were incubated with sDectin-1. After the unbound sDectin-1 was washed out with PBS, sDectin-1 and its ligand on these cells were cross-linked by Sulfo-NHS-LC-Diazirine (Thermo Scientific, Waltham, MA) according to manufacturer's instruction. Cells were lysed in T-PER Tissue Protein Extraction Reagent (Thermo Scientific), followed by the immunoprecipitation with Protein G Sepharose 4 Fast Flow (GE Healthcare). After elution with 100 mM Glycine-HCl (pH 3.0), sDectin-1-ligand complex was detected by Immunoblotting assay using HA-probe (F-7) HRP (Santa Cruz Biotechnology).

Protein samples were prepared from six discs (8 mm diameter) cut out of N. benthamiana leaves at 2 days after agroinfiltration and were homogenised in extraction buffer [10% (v/v) glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 2% (w/v) PVPP, 10 mM DTT, 1x protease inhibitor cocktail (SIGMA), 0.5% (v/v) IGEPAL (SIGMA)]. The supernatant obtained after centrifugation at 12,000 xg for 10 min was used for SDS-PAGE. Immunoblotting was performed with HA-probe (F-7) HRP (Santa Cruz Biotech) in a 1:5,000 dilution. Equal loading was checked by taking images of the stained PVDF membranes with Pierce Reversible Protein Stain Kit (#24585, Thermo Fisher Scientific).

The following antibodies were used: Gli1 (#2553S Cell Signaling, 1:1000), phospho-(Ser/Thr) AMPK Substrate (#5759 Cell Signaling, 1:1000), phospho-ACC (#3661S Cell Signaling, 1:2000), AMPK (07–181 Millipore, 1:1000), Sufu (#C8IH7 Cell Signaling, 1:1000), actin (sc-1616 SantaCruz 1:1000), tubulin (sc-8035 SantaCruz, 1:1000), laminin (sc-29012 SantaCruz, 1:1000), phospho-Serine 408 (Eurogenetec, 1:1000), HA-probe (F-7) HRP (sc-7392 SantaCruz, 1:1000), anti-Flag-M2 (F1804 Sigma, 1:1000), anti-Flag-M2 Peroxidase (A8592 Sigma, 1:5000).