Cf488a hydrazide

Cell-surface sialic acids were labeled using aniline-catalyzed oxime ligation (41 (link)). In comparison to labeling with Sambucus nigra lectin (SNA) (Vector Laboratories), which are large and have defined preferences for specific Sia linkages, chemical labeling provides a more reproducible and quantitative measurement of cell-surface sialic acids (Fig. S5). To perform this reaction, we first prepared solution A (1 mM NaIO₄ [Sigma-Aldrich] dissolved in PBS supplemented with 1 mM CaCl₂) and solution B (1 mM CaCl₂, 10 mM aniline [Sigma-Aldrich], 5% FBS and 100 μM CF633 hydrazide [Sigma-Aldrich], or CF488A hydrazide [Sigma-Aldrich] in cold PBS, pH 6.5 [Teknova]). The cells were cooled on ice and washed with cold PBS once before incubating with solution A on ice for 15 min, followed by a wash with cold PBS (pH 6.5) and incubation with solution B on ice for 40 min. The cells with labeled sialic acids were washed once with cold Opti-MEM, and their plasma membranes were labeled with CellMask Orange (Invitrogen) at a concentration of 2.5 μg/mL in Opti-MEM for 5 min at room temperature. The cells were washed again with Opti-MEM before imaging.

Sodium orthovanadate (S6508), EDTA (225658), dithiothreitol (DTT-D9779), N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (Hepes-H3375), sodium fluoride (7681-49-4), p-nitrophenyl phosphate (N1891), leupeptin (L2023), aprotinin (A4529), Protease inhibitor cocktail (P8849), ATP (A6419), Tween 20 (P1379), puromycin (P8833), RNase A (55674), Acetylcholine chloride (A6625), and CF™ 488A Hydrazide (SCJ4600015) were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). The m⁷GTP-Sepharose beads (AC-151S) were purchased from Jena Bioscience, Jena, Germany. Mouse monoclonal antibody directed against rabbit eIF4E (AB_397664) was purchased from Transduction Laboratories (Lexington, KY, USA). Horseradish peroxidase-coupled secondary antibodies (P0447) were obtained from Dako SA. Pierce ECL Plus substrate (32134) was purchased from ThermoFisher Scientific (Illkirch-Graffenstaden, France).