Semi dry transblot

Protein samples’ loading was done in order to perform 12–15% SDS PAGE. For the identification of the molecular weight of proteins, a pre-stained protein marker (Gang Nam-STAIN™, iNtRon Biotechnology, Inc., Seongnam, Korea) was utilized as a guide during the study. This protein marker included a wider range of molecular weights i.e. 10 –245 kDa. Then, transfer of proteins to PVDF membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was done through Semi Dry Trans-Blot (Bio Rad). Next to it, primary antibodies including HO-1, Nrf-2, PSD95, SYP, p-JNK and β-actin (Table 1) were applied at 4 °C overnight for the detection of various proteins after which, 5% (w/v) skim milk for blocking the membranes from the attachment of non-specific proteins was used. It was followed by rinsing the blots and incubating them at room temperature with anti-mouse secondary antibody for 2 h. Visualization of membranes was done using ECL and the achieved results were developed on X-ray films that were scanned later.Table 1

List of primary and secondary antibodies with catalog numbers.

Table 1

S.#	Antibodies Name	Catalogue #
1	Anti–HO–1	sc-136960
2	Anti-Nrf-2	sc-365949
3	Anti-PSD95	sc-71933
4	Anti-SYP	sc-17750
5	Anti-p-JNK	sc-6254
6	Anti-β actin	sc-47778
7	Goat anti-mouse (IgG-HRPs) secondary antibodies	W4028

The western blot procedure was carried out as previously described by Farooq et al., (2021) (link). All brain tissues were homogenized, centrifuged and then loaded to perform 12–15% SDS PAGE. To record the desired protein's molecular weight; a pre-stained protein marker (Gang Nam-STAIN™, iNtRon Biotechnology, Inc., Seongnam, Korea) that covers a broad range of molecular weights, i.e., 10–245 kDa range was used throughout the study. It was followed by the transfer of proteins to PVDF membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using Semi Dry Trans-Blot (Bio-Rad). A mouse derived primary monoclonal antibodies were applied at 4 °C overnight (1:1000 in TBST) to detect different targeted proteins (HO-1, COX-2, Nrf-2, TNF-α, NF-κB, and β-actin). The binding of non-specific proteins was reduced by using 5% (w/v) skim milk which blocked the membranes. The blots were rinsed, and then incubated for two hours at room temperature with horseradish-peroxidase-conjugated goat anti-mouse (IgG-HRPs) secondary antibody (1:2000 in TBST). The bands of the western blot were visualized using enchanced ochemiluminescence solution, a detection reagent according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). Results obtained were developed on the X-ray films, which were later scanned.

Protein bands were electrophoretically transferred from the SDS-PAGE gel onto a 0.2-μm nitrocellulose membrane using a semi-dry transblot (Bio-Rad) at a constant current of 120 mA for 60 minutes. After the transfer, unbound sites on the nitrocellulose membrane were blocked for 1 hour at room temperature with blocking solution (1% bovine serum albumin, BSA-TBST solution; Tribioscience Inc., Palo Alto, CA, USA) and washed 3 times in TBS with 0.05% Tween 20 (Alfa Aesar, Haverhill, MA, USA) for 5 minutes. Thereafter, the membrane was cut into strips, and each strip was incubated with individual serum samples diluted five-fold.
Immunodetection was performed with alkaline-conjugated monoclonal rabbit anti-human IgG (1:10,000), IgA (1:500), and IgM (1:500) antibodies for 30 minutes (Bethyl Laboratories Inc., Montgomery, TX, USA). Substrate development was performed using alkaline phosphatase buffer, 0.03% nitroblue tetrazolium (Thermo Fisher Scientific Inc., Rockford, IL, USA) and 0.015% 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Thermo Fisher Scientific Inc.). The MW of antigenic proteins was measured using a protein standard (Bio-Rad).