Ecl plus detecting reagents

Western blots were performed following our previous protocols 19 (link). 293T cells were transfected with RAB37-FLAG or vector control. At 48 h after transfection, the whole protein extracts were extracted from the cells and used for SDS-PAGE. The gels were transferred onto a 0.45 μm PVDF membrane (Cat# NK0414, Roche Diagnostics, Indianapolis, IN, USA). The membranes were blocked with 5% skim milk powder (Cat# 1172GR100, BioFroxx Gmbh) in TBST and incubated with the primary antibody overnight at 4 °C followed by the horseradish peroxidase-conjugated secondary antibody at room temperature for at least 1 h. ECL Plus detecting reagents (Millipore, Billerica, MA, USA) were used to visualize the protein bands. ImageJ (version J2, NIH, Maryland, US) was used to analyze band intensity values.

HEK293T cells transfected with expression vectors were lysed in RIPA lysates (50 mM Tris pH7.4, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, 1% Protease inhibitors). Cocktail (Roche) was used as protease inhibitor. After centrifuged for 10 min at 4 °C, the supernatant was used for Western blot analyses. Western blots were performed according to routine protocols. The protein extracts from HEK293T cells or mouse tissues were separated using 15% SDS-PAGE and transferred onto a 0.45 μm PVDF membrane (Cat# NK0414, Roche Diagnostics, Indianapolis, USA). The membranes were blocked with 5% non-fat dried milk in TBST (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% Tween-20) and incubated with the antibodies overnight at 4 °C, followed by the horseradish peroxidase-conjugated secondary antibody. The protein bands were visualized by incubating membranes with the ECL Plus detecting reagents (Cat# WBKLS0500, Millipore, Billerica, USA).