Rb44L1 Peptide Induces Apoptosis Signaling

B16F10-Nex2 cells (10⁶) were incubated with 0 and 130 μM of Rb44L1 peptide for different times (1, 3, 6, 8, and 24 h). After incubation, cells were washed in PBS and lysed with 300 μL of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8 at 25°C, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Proteins from whole cell extracts were analyzed by Western blotting as previously described (20 (link)). The following primary, highly specific monoclonal antibodies, were used: rabbit anti-Bcl-2, -Bcl-xl, -Bax, -caspase-9 and cleaved caspase-9, -caspase-3 and cleaved caspase-3, -Parp and cleaved Parp, and -GAPDH (for total protein loading control), with secondary anti-rabbit IgG conjugated with horseradish peroxidase (HRP). All antibodies were purchased from Cell Signaling Technology (Beverly, MA) except for anti-GAPDH, acquired from Sigma-Aldrich (St. Louis, MO). Immunoreaction was revealed using the Luminata™ Forte solution (Millipore, Billerica, MA) and images were acquired using Uvitec Cambridge (Cambridge, UK). The molecular mass of each protein was estimated based on a pre-stained protein standard (Spectra Multicolor, ThermoScientific, Waltham, MA). Full-length Western blotting membranes are displayed in Figure S1.