Irdye 800cw conjugated goat anti mouse antibody

Cells were seeded in 6 well plates (Costar; 250000 HepG2 cells/well and 125000 A549 cells/well) 1 day before incubation with macrophages. After the incubation, proteins were extracted and PARP-1 and caspase-3 protein abundance was assessed by western blotting as described previously [13 (link)]. Primary antibodies are rabbit anti-caspase-3 antibody (Cell Signaling, #9662) and mouse anti-PARP1 antibody (BD Pharmingen, #551025). Primary antibodies mouse anti-β-actin (Sigma, #A5441) or mouse anti-α-tubulin (Sigma, # T5168) were used for normalization. IRDye 800CW-conjugated goat anti-rabbit antibody (H + L; Licor, #926-32211), IRDye 800CW-conjugated goat anti-mouse antibody (H + L; Licor, #926-32210) and IRDye 680LT-conjugated goat anti-mouse antibody (H + L; Licor, # 926–68020) were used as secondary antibodies. Quantitative analysis of fluorescence intensity was measured using the Odyssey Classic Infrared Imaging System (Licor).

The extraction, separation, and detection of melanogenesis-related proteins, including MITF, tyrosinase, TRP-1, and TRP-2, were performed according to our previous method (Alehaideb et al., 2021 (link)), with modifications. Briefly, after 72 h of incubation, the untreated and treated cells were trypsinized, collected, and rinsed twice with PBS. The cell pellets were extracted once with NP40 buffer, and the protein concentrations were determined using a Qubit^® protein assay kit (Invitrogen). Cell lysates (150.0 μg) were loaded on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were detected and quantified using primary mouse monoclonal antibodies directed against melanogenesis-related proteins including tyrosinase, TRP-1, TRP-2, and MITF. Mouse monoclonal (clone 236–10501, #A11126, Thermo Fisher Scientific) and rabbit polyclonal (#ab18251, Abcam, Cambridge, MA) anti-α tubulin antibodies were loading controls. Infrared fluorescent dye IRDye^® 800CW-conjugated goat anti-mouse antibody (#926–32210) and IRDye^® 680RD-conjugated goat anti-rabbit antibody (#926–68071) were used as secondary antibodies (LI-COR Biosciences, Lincoln, NE). The blots containing the targeted proteins were scanned on the LI-COR Odyssey CLx, and the protein expression levels were analyzed using ImageJ software.