Luminata forte solution

Western blotting was performed as described previously (Khan et al., 2014 (link)) using an anti-GR antibody (H-300, Santa Cruz Biotechnology) at 1:1000 dilution, and visualized using Luminata™ Forte solution (Millipore, Billarica, MA, USA) with exposure to X-ray film (Fisher Scientific, Loughborough, UK).

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.

Rb9 and Rb10A1 peptides were diluted at 10 μg/10 μL in milli-Q water and applied on nitrocellulose membranes. Recombinant MIF and CD74 (Abcam, UK) were applied onto the nitrocellulose membranes at 50 nM and incubated overnight at 4°C. After washing, membranes were incubated with anti-MIF or anti-CD74 for 1 h at 37°C followed by several washes and anti-rabbit and anti-mouse IgG-HRP antibody incubation for 1 h at 37°C. Immunoreactivity was determined using the Luminata™ Forte solution (Millipore, Billerica, MA) and images were acquired by Uvitec Cambridge (Cambridge, UK). Some other peptides were also evaluated with negative reactivity (data not shown). This protocol was adapted from previous studies (31 (link), 32 (link)).