Ab120981

Cells were treated with 100 ng/mL of murine IFNγ (Peprotech, 315-05) for 1 h for molecular assays or longer for Seahorse and NOS activity assays (as described below). For PJ34 treatment, the cells were pre-treated with 20 µM PJ34 (Abcam, ab120981) for 1 h (BMDM, THP1) or 2 h (iBMDM) prior to stimulation. For veliparib treatment, iBMDMs were treated with 10 µM veliparib (MedChemExpress, HY-10129) for 2 h prior to stimulation. For doxycycline (Dox) induction, cells were treated with 1 μg/mL of Dox (Sigma-Aldrich, D9891) for 24 h prior to additional treatments.

A total of 30 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was incubated overnight with primary antibody after blocking with 5% skim milk. The primary antibodies were provided by Abcam Company, including anti-ERK (ab32537, 1 : 1000), anti-phospho-ERK (ab201015, 1 : 1000), anti-Nrf2 (ab62352, 1 : 1000), anti-polyadenosine-diphosphate-ribose polymerase (PARP) (ab120981, 1 : 1000), anti-GAPDH (ab8245, 1 : 1000), anti-HO-1 (ab52947, 1 : 1000), and anticatalytic subunit of glutamylcysteine ligase (GCLC) (ab207777, 1 : 1000). Membranes were subsequently incubated with appropriate HRP-conjugated secondary antibody at room temperature for 1 h. The bands were visualized using an enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA, USA) and were scanned by a luminescence image analyzer (Fuji Film LAS-4000, Japan). The bands were measured with Image Gauge software.