Anti usp48

Consecutive sections (5 μm thick) of each formalin-fixed paraffin-embedded (FFPE) tissue block were subjected to immunohistochemical staining. Briefly, the slides were deparaffinized, re-hydrated, and then immersed in distilled water with 3% hydrogen peroxidase in methanol for 20 min to suppress endogenous peroxidase activity. Sections were incubated with primary antibodies of anti-USP48 (1:200, Abcam), anti-BRAF (1:200, Abcam), anti-p-Erk1/2 (1:200, Abcam), and anti-ACTH (1:200, Abcam) at 4 °C overnight. Subsequently, an ABC staining kit (Vector Laboratories) was applied to the sections, and signals were developed with diaminobenzidine (DAB) chromogen (Vector Laboratories). The analysis of our immunohistochemical stainings is based on the evaluation method according to ref.^{38 (link)}.

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.