Jak1 6g4

Cells were lysed in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, 0.1% Triton X-100, and protease inhibitor (cOmplete; Roche). 20 µg of total protein extracts were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Optitran BA-S 85 Reinforced NC; Whatman). The membrane was incubated in PBS supplemented with 0.4% Tween-20 and 5% dry milk for 1 h. Primary antibodies recognizing Jak1 (6G4; Cell Signaling Technology), Jak2 (D2E12; Cell Signaling Technology), Stat1 (polyclonal; Cell Signaling Technology), Phospho-Stat1 (58D6 [Tyr701]; Cell Signaling Technology), ERAP1 (6H9), MECL (K6512), LMP2 (polyclonal; Abcam), LMP7 (polyclonal; Abcam), or β-Actin (C4; Santa Cruz Biotechnology, Inc.) were diluted in PBS/0.1% Tween-20 and 2.5% dry milk and were applied overnight at 4°C. The membrane was washed three times with PBS/0.1% Tween-20 followed by 1-h incubation with the secondary Ab (0.2 µg/ml goat anti–rabbit IgG H&L chain–specific peroxidase conjugate, rabbit anti–mouse IgG H&L chain–specific peroxidase conjugate, or rabbit anti–goat IgG H&L chain–specific peroxidase conjugate; EMD Millipore). The ECL Prime Western Blotting Detection kit (GE Healthcare) was used for detection. Membranes were analyzed with the Fusion FX System using FusionCapt Advance FX7 Software (Peqlab).