Immunoblotting Analysis of Signaling Proteins

Proteins (20 μg) were resolved on 10% SDS-PAGE and immunoblotted with the following antibodies: anti-S1PR₁ (1:1,000; Immunological Sciences, Cat. N. AB-83739); anti-S1PR₅ (1:1,000; Immunological Sciences, Cat. N. AB-83741); anti-phospho-AKT (1:1,000; Immunological Sciences, Cat. N. AB-10521); anti-AKT (1:1,000; Cell Signaling Cat. N. #2920), anti-phospho-ERK (1:1,000; Immunological Sciences, Cat. N. AB-82379); anti-dopamine- and cAMP-regulated protein 32 (DARPP-32; 1:1,000; Cell Signaling, Cat. N. #2302); anti-brain derived neurotrophic factor (BDNF; 1:1,000; Santa Cruz, Cat. N. sc-546).
For LC3 and Beclin1 analyses, protein lysates (20 μg) were resolved on a 12% SDS-PAGE and immunoblotted with anti-LC3 (1:1,000; Novus, Cat. N. NB100-2331) and anti-Beclin1 (1:1,000; Santa Cruz, Cat. N. sc-11427) antibodies. For protein normalization, anti-Actin (1: 5,000; Sigma Aldrich, Cat. N. A5441) and/or anti-Cyclophilin (Abcam Cat. N. ab16045) were used. Immunoblots were then exposed to specific HRP-conjugated secondary antibodies (Santa Cruz, Cat. N. sc-2004 and sc-2005). Protein bands were visualized by ECL Plus (GE Healthcare) and quantitated with Image Lab Software (Bio-Rad Laboratories). Cell lysate from HUVEC, treated with the autophagy inductor TAT-D11 (10 μM; Shoji-Kawata et al., 2013 (link)) was used as positive control.