Protein expression analysis
Protein isolation: For total protein extraction, hippocampal and
cortical samples were homogenized in RIPA buffer (150 mM NaCl, 20 mM
Tris-HCl, 1mM EGTA, 1mM Na2EDTA, 1%sodium deoxycholate,
1% NP-40, pH 7.4) supplemented with 1% protease inhibitor cocktail, 1
mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium fluoride (NaF),
and 2 mM sodium orthovanadate (Na3VO4)
(Sigma aldrich). The samples were centrifuged twice for 15 min each at
15,000 rpm at 0°C. The supernatant was removed and the protein
concentration was determined using Bicinchoninic Acid Assay (BCA) kit
(Pierce) and a microplate reader at 562 nm (Bio-Rad).
Western blot : The immuno‐related procedures used comply with the
recommendations made by the British Journal of Pharmacology (Alexander
et al., 2018). Equal amount of proteins was loaded along with dual
colour ladder (Bio-Rad) on 12-15% polyacrylamide gels and transferred
onto Polyvinylidene fluoride (PVDF) membranes. Membranes were blocked
with 3% bovine serum albumin in phosphate buffered saline with 0.1%
tween-20 for 2 hrs at room temperature; followed by primary antibodies
incubation including human anti-KAT II (1:700; abcam Cat# ab83918,
RRID: AB_2219600), human anti-KMO (1:700; abcam Cat# ab130959, RRID:
AB_11156090), human anti-PNPO (1:1000; Cat# ab83875, RRID:
AB_1861751) diluted in phosphate buffered saline with 0.1% tween-20
for 4ºC overnight and subsequently HRP conjugated secondary antibodies
incubation (1:1000, Santa Cruz Cat# sc-2004, RRID: AB_631746) diluted
in same buffer for 2 hrs at room temperature. Chemiluminescence signals
were generated using Super Signal West Pico Chemiluminescent Substrate
(Thermo Scientific) and images were captured on a Bio-Rad Chemi Doc MP
system. Band intensities were quantified by ImageJ software. Protein
expressions were normalised with respect to loading control
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
ELISA: Protein expression of Indoleamine 2,3-dioxygenase (IDO)
enzyme was performed by ELISA using commercially available kits
(Catalogue number: DY6030-05, R & D Systems, USA) according to
manufacturers’ instruction. The experiment was performed in triplicates.
Optical density was determined in a multimode reader (Synergy HTX,
Biotek, USA) at 450 nm and wavelength was corrected as 540 nm
(Nürnberger et al., 2019).