Anti p c fos

Equal amounts of protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Merk, Darmstadt, Germany). I-proteasome subunits were analyzed by immuno-blotting using anti-mouse anti-β5i/LMP7, anti-β5, anti-β1i/LMP2 (Abcam, Cambridge, UK) and anti-β1, anti-β2i/MECL-1, anti-α4 (self-made), and AP1 components, anti-(p)-cJun, anti-(p)-cFos, anti-cFos, anti-(p)-ATF-2, anti-ATF-2 (Cell signaling, Leiden, Netherlands), and anti-cJun (Santa Cruz Biotechnologie, Dallas, Texas, USA) antibodies combined with a secondary polyclonal mouse-anti-rabbit-IgG antibody conjugated to horseradish peroxidase (Dianova, Hamburg, Germany). Anti-β-actin antibody (Cell signaling, Leiden, Netherlands) was used as loading control.

Cell and tumor tissues were lysed in RIPA lysis buffer (Sigma) supplemented with complete protease inhibitor cocktail (Roche). The resultant protein extracts were subjected to western blotting using a standard protocol. The following primary antibodies were used in this study: anti-actin (1:5000, Abcam); anti-USP48 (1:1000 Abcam); anti-GAPDH (1:1000, Abcam); anti-BRAF (1:1000, Abcam), anti-Erk1/2, anti-p-Erk1/2 anti-c-Fos (1:1000, Cell Signaling Technology), anti-c-Jun (1:1000, Cell Signaling Technology), anti-Nur77 (1:1000, Abcam), anti-p-c-Fos (1:1000, Cell Signaling Technology), anti-p-c-Jun (1:1000, Cell Signaling Technology), and anti-p-Nur77 (1:1000, Cell Signaling Technology). Uncropped images of the blots are provided in Supplementary Figure 12.

Western blot experiments were performed to analyze the effect of HPE_DMSO on phosphorylation of Extra-regulated kinase 1/2 (ERK 1/2). Cells, untreated and treated for 5 min, 1 h, 4 h, and 24 h with HPE_DMSO, were washed with phosphate buffer saline (PBS) and lysated by protein extract kit (Active Motif, Carlsbad, CA, USA) in accordance with manufacturer’s instructions. Extracts were resolved on Mini-protean TGX precast gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PDVF membranes (Bio-Rad Laboratories) and probed with specific antibodies in accordance with the manufacturers’ instructions. Rabbit antibodies anti-ERK 1/2 and anti-p-ERK 1/2 (MyBioSource, San Diego, CA, USA) were used at 1:1000; rabbit antibodies anti-c-Fos and anti-p-c-Fos (Cell Signaling Technology, Danvers, MA, USA) at 1:500 and mouse anti-actin (Merck Life Science) at 1:1000. The secondary antibodies HRP-conjugate, both anti-rabbit and anti-mouse (Immunological Sciences, Rome, Italy) were used at 1:5000. The blots were revealed by ECL detection system (Advansta, Menlo Park, CA, USA). Image acquisitions were performed by ChemiDoc Instrument (Bio-Rad Laboratories).