Sc 651

Using a 7% SDS-polyacrylamide gel, electrophoresis was performed to analyze the protein from cell lysates and subsequently electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was treated with 5% non-fat milk for 1 h to block non-specific binding and probed with a rabbit anti-human polyclonal iNOS antibody (sc-651; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a final dilution of 1:1,000. The primary antibodies were detected using biotin-rabbit anti-mouse immunoglobulins G, A and M (heavy and light chains; Zymed, San Francisco, CA, USA) and alkaline phosphate-conjugated streptavidin, and were visualized using 4-nitro blue tetrazolium chloride or 5-bromo-4-chloro-3-indolyl-phosphate substrate (Promega, Madison, WI, USA).

The C6 cell lysates (20 µg) were electrophoresed and transferred to the nitrocellulose membrane. Nitrocellulose membranes, blocked in 5% of non-fat milk in PBS 0.1% Tween-20, were probed with mouse monoclonal anti-β actin antibody (antibody dilution 1:5000) (A5316 Sigma, MO, USA), rabbit polyclonal anti-VEGF anti-NOS-2 antibodies (antibodies dilution 1:200) (sc-152, sc-651, respectively, both purchased by Santa Cruz biotechnology, CA, USA), mouse monoclonal anti-HIF-1α, anti-MMP-9, anti-MMP-2, anti-NOS-1 antibodies (antibodies dilution 1:200) (sc-5354, sc-393859, sc-13595, sc-5302, respectively, all purchased by Santa Cruz biotechnology, CA, USA), then incubated in the presence of specific enzyme conjugated IgG horseradish peroxidase. Immunoreactive bands were detected by ECL system (Amersham Int., Buckunghamshire, UK) and analyzed by densitometry. Densitometric values, expressed as Integrated Optical Intensity (IOI), were estimated in a CHEMIDOC XRS system by the QuantiOne 1-D analysis software (BIORAD, Richmond, CA, USA). Values obtained were normalized based on densitometric values of loading control β-actin.

Acetic acid, disodium hydrogen phosphate, reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), Potassium chloride, Ammonum chloride, Sodium dihydrogen phosphate, and Bovine Serum Albumin (BSA) were procured from Sisco Research Laboratories (SRL; Mumbai, India). 1-Methyl-4-phenyl-1,2,3,6-tetra hydropyridine (MPTP), Normal Goat Serum (NGS) from Sigma–Aldrich (St. Louis, MO, United States). Protein estimation kit by Bradford GeNeiTM, hydrogen peroxide (H₂O₂), and potassium dichromate were purchased from Merck (Darmstadt, Germany), Sodim dodecyl sulfate (SDS), Thiobarbituric acid (TBA), Griess reagent and DABCO were procured from HiMedia (Mumbai, India). Sodium nitrite and Paraformaldehyde were purchased from Lobachemie, India. Primary antibodies for TH (SC-25269), iNOS (SC-651), Glial Fibrillary Acidic Protein (GFAP) (SC-33673) and Intercellular Cell Adhesion Molecule (ICAM) (SC-8439) were procured from Santa Cruz, Biotechnology (Santa Cruz, CA, United States) and the primary antibodies for TNF-α (ab1793), NF-κB (ab16502), DAT (ab111468), and pAkt1 (ab81283) were purchased from Abcam Life Science, Biogenuix Medsystems, Pvt. Ltd. (New Delhi, India), secondary fluorescent tagged antibodies for IHCCy2-conjugated and cy3-conjugated were procured from Merck Millipore and Chemicon respectively.

Chlorogenic acid, MPTP, and normal goat serum (NGS) were bought from Sigma–Aldrich (St. Louis, MO, United States). Acetic acid, ammonium chloride, bovine serum albumin (BSA), potassium chloride, disodium hydrogen phosphate, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and sodium dihydrogen phosphate were procured from Sisco Research Laboratories (SRL, Mumbai, India). Protein estimation kit by Bradford GeNeiTM, hydrogen peroxide (H₂O₂), and potassium dichromate were purchased from Merck (Darmstadt, Germany), and sodium dodecyl sulphate (SDS), thiobarbituric acid (TBA), DABCO, and Griess reagent were obtained from HiMedia (Mumbai, India). Paraformaldehyde and sodium nitrite were bought from Lobachemie, India. Primary antibodies for TH (SC-25269), iNOS (SC-651), and glial fibrillary acidic protein (GFAP; SC-33673) were purchased from Santa Cruz, Biotechnology (Santa Cruz, CA, United States), and the primary antibodies for TNF-α (ab1793) and NF-κB (ab16502) were obtained from Abcam Life Science, Biogenuix Medsystems Pvt. Ltd. (New Delhi, India), and secondary fluorescent-tagged antibodies such as Cy2 conjugated and Cy3 conjugated for immunohistochemistry were bought from Merck Millipore and Chemicon, respectively.