Caspase 3

HaCaT and A549 cells were treated with gZnNPs (0, 25, and 35 μg/ml) for 24 h, and after exposure, the cell lysate was prepared in RIPA buffer (ab156034). The cell lysate was centrifuged at 13000 rpm, 4°C for 30 min, and the supernatant was used for protein expressions. The concentration of protein was evaluated by the Bradford method [11 (link)]. Protein (20 μg) was migrated on the gel and transferred to a PVDF membrane (Bio-Rad, Laboratories Inc., Berkeley, CA, USA). The PVDF membrane was incubated with different mouse monoclonal antibody against β-actin (1 : 12000 dilutions, Abcam, Cambridge, UK), Bcl2 (1 : 500 dilutions, Santa Cruz), Bax (1 : 1000 dilutions, Antibodies-online), Caspase-3 (1 : 500 dilutions, Cayman), and TNF-α (1 : 500 dilutions, Santa Cruz) for 24 h at 4°C.
Secondary antibody HRP-conjugated goat anti-mouse IgG (H + L) antibody (1 : 2000 dilutions Bio-Rad) was used. Immunoreactive bands were detected using an EZ west Lumi plus (ATTO Corporation, Tokyo, Japan), which is a chemiluminescent substrate to detect HRP on the western blotting membrane. The luminescence intensity (optical density) of the target protein bands was quantified using Lumino Graph 2 (ATTO Corporation). All protein expression levels were normalized to the levels of β-actin protein expression in each band.

Rats were sacrificed by decapitation at 4 weeks post-induction (n = 4 per group per time point). The whole brain was collected from each animal. Proteins were separated by 12% SDS-PAGE electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were then incubated with primary polyclonal rabbit antibodies against phosphorylated tau (p-tau; 1:400; Thermo Fisher Scientific), LC3BII (1:800; Novus Biologicals), NF-κB (1:800; R&D Systems), CD68/ED1 (1:500; Santa Cruz Biotechnology), ZO-1 (1:800; Santa Cruz Biotechnology), COX-2 (1:1500; Neuromics, Edina, MN, USA), Syn (1:1200; Thermo Fisher Scientific), RhoA (1:500; Upstate Biotechnology), ICAM-1 (1:500; Novus Biologicals), AGT (1:1200; Chemicon), PI3K/p85 (1:500; Thermo Fisher Scientific), STAT3 (1:800; Cayman Chemical), p-AKT (1:800; Abcam), ACH (1:500; Sigma), caspase 3 (1:1000; Cayman Chemical), and CHAT (1:500; Abcam) at room temperature for 12 h. The membranes were then re-probed with a rabbit anti-β-actin antibody (1:5000; Abcam). After incubation with a goat anti-rabbit secondary antibody (1:5000; Santa Cruz), the protein bands were detected using an enhanced chemiluminescence reagent.

RTCs were harvested in 10-cm² dishes after the indicated treatment. The cells were partitioned into cytosolic and nuclear fractions by using NE-PER^TM nuclear extraction reagents (Pierce, Rockford, IL, USA) with the addition of protease inhibitors, according to the manufacturer’s instructions. Antibodies against PPARγ (SC-7273), Bcl-xL/xS (SC-1041), NFAT3 (SC-13036), NFκB-p65 (SC-372), PGC1α (SC-13067), Sirt1 (SC-15404), pan-acetylated (SC-8649) and Lamin A/C (SC-6215) (1:500 Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 (#13909; 1:500, Cayman Chemical, Ann Arbor, MI, USA), GAPDH (#LF-PA0018; 1:2000, Ab Frontier, Seoul, Korea), and β-actin (#MABT523; 1:500, Millipore, Burlington, MA, USA) were included in the assay. The cell lysate (50 μg) was electrophoresed on an 8% sodium dodecylsulfate-polyacrylamide gel and then transblotted onto a Hybond-P membrane (GE Healthcare, Hong Kong SAR). The subsequent procedures were as described previously [41 (link)]. Western blot bands were quantified by using the Scion Image Software (Scion, Frederick, MD).