Immunoblotting for Neuronal Protein Expression

Immunoblotting for the expression of neuronal cell-specific proteins was performed with both induced and uninduced control cells, as previously described^{7 (link)}. Briefly, after preparing whole cell lysates using RIPA buffer (#R0278, Sigma, USA), the protein quantification was done using bicinchoninic acid (BCA) assay method, according to the manufacturer’s protocol (#00-4333-57, Pierce, ThermoScientific, USA). Protein extracts (30 μg) were subjected to SDS–PAGE using 12% Tris/HCl sodium dodecyl sulphate (SDS) gels and transferred onto PVDF membranes (Membrane Technologies, India). After blocking the membranes with 3% BSA, they were incubated with primary antibody against β-actin (1:2500, #ab8227), MAP-2 (1:1500) and TH (1:1000, #sc-73151, Santa Cruz, USA) in 1% BSA-phosphate saline buffer (PBS) overnight at 4 °C. Post incubation, membranes were washed thrice in PBST and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1/4000) (Dako, USA) for 2 h at RT. Membranes were developed with chemiluminescence detection reagent (#34580, Pierce, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Australia).