*4.5. Immunoblot Analysis*

SH-SY5Y cells (2.2 <sup>×</sup> 104 cells/mL) were pre-treated in 60 mm cell culture dishes with NE 52-QQ57 (100 nM) and left in serum-free media for 1 h. They were then stimulated with or without MPP<sup>+</sup> (1 mM) or H2O2 for 24 h, again in a serum-free media. Next, the cells were washed two times with PBS and lysed for 10 min at 4 ◦C using an RIPA lysis buffer (with protease and phosphatase inhibitors). Supernatants were collected for further investigation after the cell lysates were centrifuged at 14,000 rpm, at 4 ◦C. The protein concentration of each sample was measured and normalised using a DC Protein Assay kit (Bio-Rad). Equal amounts of proteins (20–30 μg) were loaded and separated electrophoretically in 8, 10, and 12% sodium dodecyl sulphate-polyacrylamide gels; these were then transferred to polyvinylidene difluoride membranes (Millipore; Bedford, MA, USA). The membranes were incubated overnight at 4 ◦C, with corresponding primary antibodies, GPR4 (1:500) from Novus Biologicals (Centennial, CO, USA), BCL-2 (1:1000), Caspase-3 (1:1000), cleaved Caspase-3 (1:1000), cleaved PARP (1:1000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Bax (1:1000) from Cell Signaling Co. (Boston, MA, USA), PIP2 (1:500) from Abcam (Cambridge, United Kingdom), and β-Actin (1:2000) from Sigma-Aldrich (St. Louis, MO, USA), followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; Cell signalling, MA, USA). The blots were visualised using a Biorad-ECL (Bio-Rad Laboratories; Hercules, CA, USA) and photographed. Using ImageJ (NIH) software, the pixel intensity for each band in the photographs was measured and normalised to the band intensity of β-Actin, to quantify its relative expression.
