Specific hrp conjugated secondary antibodies

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.

A549 cells and H1299 cells infected with lentivirus expressing either Scr-shRNA or GMDS-shRNA were cultured for 2 days for protein isolation. In brief, cells were washed with PBS buffer and harvested with lysis buffer (100 mM Tris-HCl, pH = 7.4 l 0.15 M NaCl; 5 mM EDTA, pH = 8.0; 1% Triton X100; 5 mM DTT; 0.1 mM PMSF) to extract total proteins which were quantified with BCA Protein Assay Kit (Pierce, Rockford, IL, USA). To perform western blot analysis, 20 μg protein samples were mixed with loading buffer. Then SDS-PAGE electrophoresis and subsequent PVDF transmembrane were performed (Amersham Biosciences, Pollards Wood, UK). Membrane was blocked with 5% milk dissolved in TBST buffer for 1 h and then incubated with primary antibodies overnight at 4 °C. Primary antibodies used here were as follows: Rabbit anti-GMDS, Novus Biological, NBP1–33424 (1:500); Rabbit anti-CDKN1A, Abcam, ab7960 (1:500); Mouse anti-DDIT3, Abcam, ab11419 (1:1000); Rabbit anti-FAS, Abcam, ab82419 (1:1000); Rabbit anti-JUN, Abcam, ab32137 (1:1000); Rabbit anti-VEGFA, Abcam, ab183100 (1:500); mouse anti-Flag, Sigma, F1804 (1:1000); mouse anti-GAPDH, Santa-Cruz, sc-32,233 (1:2000). After washing with TBST buffer for three times, specific HRP conjugated secondary antibodies from Santa Cruz were added and immunoactivity was detected with ECL-Plus kit (Amersham Biosciences, Pollards Wood, UK).