Sc 2325

Cells were washed with PBS and lysed in lysis buffer (1% SDS, 62.5 mM Tris–HCl pH 6.8, 1% Phenylmethylsulfonyl fluoride (v/v), containing 1 × protease inhibitor cocktail (Roche, 4,693,132,001) and 100 U benzonase nuclease (Novagen, 70,746–3). Subsequently, lysates were homogenized using a syringe, 4 × loading buffer was added (20% glycerol, 6% SDS, 25% Tris–HCl pH 6.8, 10% β-mercaptoethanol and bromophenol blue in MilliQ) and samples were boiled for 5 min at 95 °C. Lysates were resolved on a 7% SDS-PAGE gel and blotted to a nitrocellulose membrane. The membrane was blocked for 1 h at RT using blocking solution (3% bovine serum albumin (Roche, 10,735,094,001) and 1% non-fat dry milk (Santa Cruz Biotechnology, sc-2325) in PBS-T (0.1% Tween 20 in PBS)). Subsequently, the membrane was washed with PBS-T and stained using 1 μg/ml mouse anti-Siglec-7 (clone 194,211, R&D Systems, MAB11381) and 1:1000 rabbit anti-β-actin (clone 13E5, Cell Signaling, 4970S, RRID:AB_2223172) in 1:4 blocking solution in PBS-T overnight at 4 °C. After washing the blot with PBS-T, it was incubated with 0.2 μg/ml IRDye 800CW goat-anti-mouse IgG (Westburg, 926–32,210, RRID:AB_621842) and 0.2 μg/ml IRDye680RD goat-anti-rabbit (Westburg, 926–68,071, RRID:AB_10956166) for 1 h at RT. Finally, the blot was washed with PBS-T and imaged on an Odyssey CLx infrared imaging system (LI-COR).