Mouse monoclonal antibody against β actin

Rabbit polyclonal antibody against phospho-eIF2α (32157) and mouse monoclonal antibody against β-actin were purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibodies against, eIF2α (9722) and anti-phospho-eIF2α (3597) were from Cell Signaling Technology (Danvers, USA).

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.