Mouse anti β tubulin

Western blot analyses were performed as described below. Briefly, 16 h after transfection, HEK293T cells were treated with drugs and on the next day cells were lysed in the NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 1 mM NaF, 50 mM Tris, pH 7.6) supplemented with protease inhibitor mixture (05892791001, Roche). After centrifugation at 12,000 × g at 4 °C for 10 min, the supernatants were collected for Western blot analyses (full blots found in Supplementary Fig. 10). The primary antibodies used in this study were rat anti-HA (11867423001, Roche) with a dilution 1:3000, rabbit anti-PKCε (sc-214, Santa Cruz) with a dilution 1:1000, mouse anti-β-Tubulin (M30109, Abmart) with a dilution 1:3000.

Protein solutions from three mice in each group were denatured with 5 × loading buffer at 95 °C for 10 min and stored at − 20 °C. Twenty to forty micrograms of total protein sample were separated by SDS-polyacrylamide gel electrophoresis using a Bio–Rad protein assay and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in Tris-buffered saline/0.1% Tween buffer (TBST; 25 mM Tris–Cl, 125 mM NaCl, 0.1% Tween-20) with 5% skim milk powder at room temperature for 2 h and incubated with primary antibodies at 4 °C overnight: mouse anti-GLUT5 (Santa Cruz, sc-271055, 1:300), mouse anti-GLUT3 (Abcam, ab150299, 1:1000), mouse anti-ADAM10 (Santa Cruz, sc-28358, 1:500), mouse anti-SYK (Santa Cruz, sc-1240, 1:300), rabbit anti-phospho-SYK (Tyr525/526) (CST, 2710, 1:300), mouse anti-β-tubulin and mouse anti-β-actin (Abmart, 1:1000). The corresponding secondary antibody was incubated at room temperature for 2 h (1:4000). The results were analyzed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA).