Anti ikkα β

Lipopolysaccharide (LPS, E. coli 0111: B4, TLR4 ligand), poly(I:C) (TLR3 ligand), and CpG ODN (TLR9 ligand) were purchased from Sigma-Aldrich. ChIP Grade Protein G Magnetic Beads, Cell Lysis Buffer, Anti-Myd88, Anti-Traf6, Anti-p65, Anti-p-p65 (Ser536), Anti-Ikkα/β, Anti-p-Ikkα/β (S176/180), Anti-IκBα, Anti-p-IκBα (S32), Anti-ERK, Anti-p-ERK (Thr202-Tyr204), Anti-JNK, Anti-p-JNK (Thr183-Tyr185), Anti-p38, Anti-p-p38 (Thr180-Tyr182) and Anti-Syk and Anti-p-Syk (Tyr-525/526) were from Cell Signaling Technology. Anti-DAP12 was purchased from Abcam. Anti-β-actin, Anti-rabbit IgG-HRP, and anti-mouse IgG-HRP were from Santa Cruz Biotechnology. Anti-LaminA/C, Anti-Flag and Anti-Myc and Anti-V5 were purchased from Proteintech. Anti-Ocilrp2 (AF3370) was from purchased R&D. The NF-κB inhibitor BAY11-7082 (10 μM), Tbk inhibitor Amlexanox (10 μM), Mek inhibitor PD98059 (10 μM), PI3K inhibitor Wortmannin (5 μM), Erk inhibitor SHC772984 (10 μM), Jnk inhibitor SP600125 (10 μM), or p38 inhibitor SB203580 (10 μM), Syk inhibitor R406 (5 μM) were from Selleckchem.

Cells were rinsed with ice-cold lysis buffer (50 mM Tris, 10 mM EDTA, 1% v/v Triton-X100), supplemented with the protease inhibitor cocktail set III (80 μM aprotinin, 5 mM bestatin, 1.5 mM leupeptin, and 1 mM pepstatin; Calbiochem, San Diego, CA, USA), 2 mM phenylmethylsulfonyl fluoride and 1 mM Na₃VO₄, then sonicated and centrifuged at 13,000× g for 10 min at 4 °C. 20 μg protein extracts were subjected to SDS-PAGE and probed with antibodies for: Anti-Pgp/ABCB1 (Calbiochem), anti-MRP1/ABCC1 (Abcam, Cambridge, UK), anti-BCRP/ABCG2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-(phosphoSer473)Akt (Cell Signalling Technology, Danvers, MA, USA), anti-Akt (Cell Signalling Technology), anti-(phosphoSer176/180)IKKα/β (Cell Signalling Technology), anti-IKKα/β (Cell Signalling Technology), and anti-IkBα (Santa Cruz Biotechnology Inc.), followed by a peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). The membranes were washed with Tris-buffered saline-Tween 0.1% v/v solution, and the proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). To check the equal control loading in lysates, samples were probed with an anti-β-tubulin (Santa Cruz Biotechnology Inc.) antibody.