α ubiquitin p4d1

For direct immunoblot experiments, cell lysates were prepared using the rapid boiling method (Kushnirov, 2000 (link)). Proteins were separated by SDS–PAGE (10% or 4–20% gradient gels, BioRad) followed by immunoblot analyses. Mouse monoclonal α-HA (16B12; Covance), α-GFP (Roche), α-carboxypeptidase Y (CPY) or goat polyclonal α-Mpk1 (yC-20; Santa Cruz) were used at a dilution of 1:10,000. Mouse monoclonal α-ubiquitin (P4D1; Cell Signaling), rabbit polyclonal α-phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling), and rabbit polyclonal α-Rad53 (abcam) were used at a dilution of 1:2000. Secondary goat anti-mouse (Jackson ImmunoResearch), donkey anti-rabbit (GE Healthcare), and donkey anti-goat (Santa Cruz) antibodies were used at a dilution of 1:10,000.

The following antibodies against murine antigens were used: α CD152 (UC10-4F10), isotype control antibody (A19-3), α Vα2-TCR (B20.1), α T-bet (O4-46), α CD44 (IM7), α CD45.2 (104) (BD Biosciences, Franklin Lakes, NJ, USA); α CD3 (145-2C11), α CD28 (37.51), α CD8a (53-6.7), α CD62L (MEL-14), α IFN-γ (XMG1.2) (BioLegend, Koblenz, Germany); α Fra-2 (Q20), α GAPDH (A3), α eIF4A (yN-20) (Santa Cruz Biotechnology, Dallas, TX, USA); α PDCD4 (D29C6), α eIF4G (C45A4), α eIF4A (C32B4), α FoxO1 (C29H4), α phospho-FoxO1^S253, α phospho-AKT^S473, α phospho-AKT^T308, α Akt (pan) (C67E7), α SENP3 (D20A10), α Aldolase A (D73H4), α Lamin B1, α Ubiquitin (P4D1) (Cell Signaling Technology, Danvers, MA, USA); α phospho-PDCD4^S457 (9G6) (Rockland Immunochemicals, Limerick, PA, USA); α Glutaminase (EP7212) (Abcam, Cambridge, UK). For in vivo antibody treatment of mice α CTLA-4 (UC10-4F10) were purified from hybridoma supernatants and controlled by flow cytometry. α DEC-OVA conjugates were produced as previously described.^{51 (link)}