Ab3328

For protein isolation, alveolar macrophages were washed twice with ice cold PBS and lysed with RIPA buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS). Samples were centrifuged to remove cell debris and protein concentrations were determined using standard Bradford assays. In all, 10 μg of protein lysates were separated on 10% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (162–0177, Bio-Rad, Hercules, CA, USA). The following antibodies were used: LMP2 (ab3328, Abcam, Cambridge, UK), LMP7 (ab3329, Abcam), 20S α1+2+3+5+6+7 (hereafter called α1–7; clone MCP231, ab2267, Abcam), Psmb5 (detecting β5, ab90867, Abcam), AKT (4685, Cell Signaling, Beverly, MA, USA), p-AKT (4060, pSer⁴⁷³, Cell Signaling), STAT6 (9362, Cell Signaling), p-STAT6 (9361, pTyr⁶⁴¹, Cell Signaling), IRF4 (M17, Santa Cruz, Dallas, TX, USA), Arginase 1 (H-52, Santa Cruz), iNOS (610331, BD Transduction Laboratories, Franklin Lakes, NJ, USA), IL4R (ab157162, Abcam), HRP-conjugated anti-β-actin (Sigma-Aldrich), HRP-conjugated anti-rabbit (Abcam) and anti-goat antibodies (Santa Cruz).