Genesys image acquisition software

PC346Flu1 cells were either left untreated (control) or treated with 250 mM or 400 mM carnosine dissolved in cell culture medium. After 24 h of carnosine treatment cells were lysed using radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor (Sigma‐Aldrich), vigorously vortexed then placed on ice every 10 min for a 30‐min period. The mixture was then centrifuged at 14,000 × g for 15 min at 4°C to separate the cellular debris from the lysate. The concentration of protein within the lysate was quantified using a bicinchoninic acid (BCA) assay. For western blotting analysis, an equal amount of protein (20 μg) was loaded per well. Proteins were separated using SDS‐PAGE and then transferred to a nitrocellulose membrane. Membranes were then blocked using a 5% (w/v) milk solution in TBS‐T. After blocking, membranes were probed with antibodies against SIRT3 (mouse monoclonal from Santa Cruz Biotechnology) and vinculin (rabbit monoclonal from Abcam). After exposure to primary antibodies, membranes were washed and probed with HRP‐conjugated horse anti‐mouse and goat anti‐rabbit IgG antibodies (Cell Signaling Technology). Chemiluminescent signals were then detected using Clarity Western ECL substrate (Bio‐Rad) and imaged using a Syngene G:BOX station with GeneSys image acquisition software (Syngene). ImageJ software (NIH) was then used to quantify the band intensity.

All samples were resolved by SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in 0.1% PBS-T (phosphate buffered saline with Tween-20) and probed with an anti-Firefly antibody (ab185924, Abcam, 1/5000) or anti-SNAP-25 antibody (S9684, Sigma-Aldrich, 1/2000). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary anti-rabbit (A6154, Sigma-Aldrich, 1/2000) antibodies and SuperSignal West Dura ECL substrate (Thermo Fisher Scientific). Bands were visualized on a Pxi4 imaging system using GeneSys image acquisition software (Syngene, Bangalore, Karnataka, India).
For SNAP-25 cleavage assay, intensities of the total form and cleaved form of SNAP-25 were measured with GeneTools (Philomath, OR, USA).

BV2 microglia were seeded at a cell density of 625 × 10³ cells/well in a 6-well plate overnight. The cells were treated as mentioned in Experiment 2.7.2. Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with cell lysis buffer containing PMSF and protease inhibitor on ice for 5 min. Cell lysates were collected and centrifuged at 150,000 rpm at 4 °C for 10 min to remove the cell debris. Protein concentration was quantified using the BCA protein assay kit. An equal amount of protein was separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk for 1 h and subsequently incubated with the primary antibody (COX-2 (1:1000), iNOS (1:500), or β-actin (1:1000)) at 4 °C overnight. The membrane was washed and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10,000) for 1 h. After washing, the membrane was added with ECL, and the signal was visualized using the G:BOX Chemi XX9 gel doc system and GeneSys image acquisition software (Syngene, Cambridge, UK). Band intensity was quantified using Image J software (version 1.50, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The original Western blot images were shown in Figure S1.