Maackia amurensis lectin 2 mal 2

For immunoprecipitation (IP) of sialylated proteins with MAL-II lectin, cells were lysed in cold IP buffer (0.5% NP-40, 100 mM NaCl, 5 mM EDTA, 10% glycerol, and 50 mM Tris-HCl pH 7.5) supplemented with protease and phosphatase inhibitors, and supernatant was collected as WCE after centrifugation at 20,000×g for 15 min at 4 °C. In all, 1 mg WCE was diluted with IP buffer to a final volume of 500 μl and incubated for 3 h at room temperature with 10 μg of biotinylated Maackia Amurensis Lectin II (MAL-II; Vector Laboratories). Streptavidin-agarose beads (Thermo Fisher Scientific) were then added and samples were incubated for additional 2 h at room temperature in costant rotation. Lectin-bounded sialylated proteins were collected after brief centrifugation, washed with IP buffer, eluted from beads by boiling in SDS–PAGE sample buffer 2X and resolved by SDS-polyacrylamide gel electrophoresis.

FLAG‐tagged mGBP1 and mMx1 proteins were visualized in LA‐4 cells by confocal microscopy. Cells were seeded into 8‐well chamber slides (Lab‐Tek, Nunc, St Louis, MO, USA) (cultured for 24 h in the presence or absence of 1 μg mL⁻¹ DOX), and cell surface staining was performed prior to fixation using biotinylated Maackia Amurensis Lectin II (MAL II; Vector Laboratories, Newark, CA, USA) followed by Alexa Fluor 488 streptavidin (Molecular Probes, Eugene, OR, USA) in cold 0.05 m Tris–HCL, 0.15 m NaCl buffer, pH 7.2 containing, 10 mm CaCl₂ and 1% BSA. Cells were then fixed in phosphate‐buffered saline (PBS) containing 4% (vol/vol) paraformaldehyde before permeabilization in PBS containing 5% (wt/vol) bovine serum albumin, 5% (vol/vol) FCS and 0.1% (vol/vol) Triton X‐100. FLAG was detected using an anti‐FLAG antibody conjugated to Alexa Fluor 647 (L5; BioLegend) followed by additional amplification of the signal using goat anti‐rat immunoglobulin conjugated to Alexa Fluor 647 (Invitrogen, Waltham, MA, USA). Prior to visualization, cells were counterstained with VECTASHIELD Antifade Mounting Medium containing 4′,6‐diamidino‐2‐phenylindole (Vector, Burlingame, CA, USA). Images were acquired with a Zeiss LSM780 microscope and were processed using FIJI ImageJ software (Version 2.1.0/1.53c, National Institutes of Health, Bethesda, MA, USA).