Pna biotin

Abs specific for CD19, CD3e, CD4, PD-1, APRIL, CD11c, IL-12p40, Ter119, CD11c, MHCII IA/IE, TACI were purchased from Biolegend; GL7, FAS, CXCR5, CD138, B220, IgG, Siglec, CD19, CD8, CD45RA, Annexin V, and Streptavidin-HRP from BD Biosciences; GR-1, CXCR4, CD3e, CD49b, CD40, CD86, CD80 from eBiosciences; BCMA from R&D Systems. IgM (B7.6) and anti-Fc (2.4G2) were purified from hybridomas. Live/Dead Pacific Orange, Fixable Blue dyes, anti-IgG-Alkaline Phosphatase, anti-IgM- Alkaline Phosphatase, Streptavidin were purchased from Invitrogen/Life Technologies; SDF-1 from Shennendoah Biotechnology; anti-IgG2- Alkaline Phosphatase from Southern Biotech; p-Nitrophenyl Phosphate tablets, 3-amino-9-ethylcarbazole tablets, PNA-biotin, and lipopolysaccharide from Sigma; anti-IgGFc from Jackson Immunoresearch; Brefeldin A from LC Laboratories. IsdB peptide (AA 40-613) was a gift from Dr. Benfang Lei (Montana State University). Pokeweed mitogen was a gift from Dr. Shane Crotty (La Jolla Institute for Allergy & Immunology).

Cell lysates were harvested in RIPA buffer (Sigma) containing Complete Protease Inhibitor (Roche) and Phospho Stop (Roche) on ice and run on SDS-PAGE gels. PVDF membranes were stained for galectin-3 (Abcam, ab53082, 1:500, or BioLegend, M3/38, 1:1000), galectin-8 (Abcam, ab69631, 1:500), and α-tubulin (Cell Signaling, 2125, 1:1000). Lectin blotting for glycans was performed using PNA-Biotin (Sigma) followed by detection using the ABC Elite Kit (Vector Labs, PK-6100) or PNA-Peroxidase conjugates (Sigma). Ponceau S solution (Thermo) was used for total protein detection following transfer to PVDF membranes.
For liver homogenate western blots, mice bearing 393M1 flank tumors or no tumors were perfused by intracardiac injection of 20mL of phosphate buffered saline. Livers were then excised and 50mg portions were added to gentleMACS M tubes (Miltenyi Biotech) in 4.5mL of RIPA (Sigma) with Complete Protease Inhibitors (Roche). Tissue was dissociated using the gentleMACS Octo Dissociator (Miltenyi Biotech) and run on polyacrylamide gels as described above.