Aal agarose beads

Lyophilized, iTRAQ-labeled samples (200 µg peptide) were reconstituted with 1 mL lectin binding buffer (20 mM Tris, 0.3 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4) and incubated with 2 mg AAL agarose beads (Vector Laboratories, Burlingame, CA, USA) via rotation at room temperature for 1 h. After washing with lectin binding buffer three times and 500 µL ddH₂O twice, AAL pull-down peptides were transferred to a new tube and eluted using 10% AA/30% ACN with shaking at room temperature for 10 min. Eluted peptides were re-lyophilized and dissolved in 50 mM sodium phosphate buffer, pH 7.5, supplemented with 90% H₂¹⁸O and 40 U (1 unit/µL) N-glycosidase F (PNGase F; Roche Applied Science, Mannheim, Germany) and incubated at 37 °C with slight shaking for 20 h. Deglycosylated peptides were desalted with 40 µL C18 resin (source 15RPC, GE Healthcare, Björkgatan, Sweden) and lyophilized for further analysis using 2D-SCX/RP-LC-MS/MS.

AAL-agarose pull-down assays were performed to examine the levels of O-fucosylation in WT SPY and spy mutants. For the pull-down assays, Myc-SPY and Myc-spy protein amounts in different N. benthamiana tissues were adjusted to be the same by adding non-infiltrated leaf tissues. Then, 250 mg of agro-infiltrated leaf tissue was homogenized in 1.5 mL of Extraction Buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5% Triton X-100, 2.5 mM 2-mercaptoethanol, 20 µM MG-132, 1× Complete EDTA-free protease inhibitors (Sigma-Aldrich) on ice and centrifuged at 15,000×g for 10 min at 4 °C. After this, 20 µL of each protein extract was mixed with 30 µL of 2× Laemmli Sample Buffer (LSB) (Bio-Rad) for input analysis. To 1.5 mL of extract, 20 µL of AAL-agarose beads (Vector Labs, AL-1393-2, 2 mg lectin/mL) were added and incubated with rotation for 1.5 h at 4 °C. The beads were washed with extraction buffer, four times with TBST-500, and once with 20 mM Tris-HCl, pH 7.5, and were boiled in 50 µL of 2 × LSB for gel blot analysis.
To examine the differential binding affinity of SPY and spy mutants to RGA, FLAG-RGA was expressed alone or co-expressed with Myc-SPY or Myc-spy mutant proteins in N. benthamiana, and subsequent co-IP assays using anti-cMyc rabbit antibody conjugated agarose beads (Sigma-Aldrich, A7470) were performed as described^{17 (link)}.