Swga agarose

In all, 3 × 10⁷ Ramos B cells were lysed in 400 μl IP lysis buffer and subjected to immunoprecipitation with 50 μg biotin-phalloidin (Sigma, P8716) at room temperature for 12 h, followed by incubation with 50 μl streptavidin agarose resin (Thermo Fisher Scientific, 20347) at room temperature for 2 h and then spin-down at 1,000g at 4 °C for 1 min. The supernatant was collected and used as the F-actin non-enriched fraction. Biotin-phalloidin-/streptavidin agarose-bound proteins were resuspended in 400 μl IP lysis buffer and then used as the F-actin-enriched fraction. To detect the O-GlcNAcylation levels of Lsp1 within each fraction, 50 μl sWGA agarose (Vector Laboratories, AL-1023S) was added as described above.

Secreted GspB736flag was purified from M99 culture supernatants (14 Liters) as previously described [10 (link)]. In brief, M99 strains PS3309 and PS3540 were grown overnight in THB and cells removed by centrifugation. Proteins secreted into the culture media were precipitated overnight in ammonium sulfate (NH4)₂SO₄ (25% final concentration), recovered by centrifugation and reconstituted in Tris buffered saline (TBS: 50 mM Tris pH 7.5, 150 mM NaCl). Glycosylated GspB736flag was subsequently purified from TBS under native conditions by affinity chromatography using sWGA agarose (Vector) and eluted in 300 mM GlcNAc. GspB736flag fractions were pooled, concentrated by ultrafiltration using an Amicon Ultra centrifugal filter device (100 kDa cutoff) and reconstituted in dH₂O until further analysis.

Mouse liver nuclear lysates (100 μg per sample) were incubated for 1 h (4 °C) with protein A/G agarose beads (sc-2003; Santa Cruz Biotechnology), then centrifuged. The resulting supernatants (cleared extracts) were transferred to new tubes and incubated overnight (rotating; 4 °C) with 40 μL succinylated wheat germ agglutinin (sWGA)-agarose (Vector Laboratories, Burlingame, CA, USA). After washing four times with PBS containing 0.2% NP-40, proteins were eluted from the beads by boiling for 10 min in NuPage LDS sample buffer, and resolved by SDS-PAGE. The captured proteins were analyzed by immunoblotting as described above.