Anti irak1 antibody

Pituitaries (n = 6 per group) and inguinal lymph nodes were homogenized in lysis buffer (Abcam, Cambridge, MA, USA) by mortar and pestle in liquid nitrogen. Lysates were centrifuged to remove debris and quantified by a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Cleared lysates (60 μg/lane) were separated by a reducing SDS–PAGE and transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin in PBST (PBS containing 0.05% Tween-20 (VWR-Amresco, Radnor, PA, USA)), the membranes were incubated with an anti-IRAK1 antibody (1:1000, Cell Signaling, Danvers, MA, USA), followed by a peroxidase-conjugated anti-rabbit secondary antibody (1:20,000, Jackson ImmunoResearch, West Grove, PA, USA). The blots were developed by adding a chemiluminescent substrate (Thermo Scientific), and chemiluminescence images were recorded by a CCD imaging system (Hansor, Taichung, Taiwan). The images of IRAK1 and β-actin blots on the same membrane were quantified with the Image J software (version 13.06 for Mac OS X, 64 bit, free software, National Institutes of Health, Bethesda, MD, USA (accessed on 25 August 2021)). Signal intensities of IRAK1 were divided by the intensities of β-actin to obtain normalized IRAK1 expression.

RPMI-1640, FCS, phalloidin Alexa Fluor 546 and goat anti-mouse Alex Fluor 488 were obtained from LifeTechnologies (Darmstadt, Germany). PBS was from Biochrom GmbH (Berlin, Germany). PMA was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). LTA was from InvivoGen (Toulouse, France). DAPI was from AAT Bioquest (Sunnyvale, USA). IKK inhibitor XIII was obtained from EMD Millipore Corporation (Billerica, USA). zVAD-fmk was obtained from AdipoGen (Liestal, Switzerland) Salmonella minnesota LPS was from Enzo Life Sciences (Lörrach, Germany). Human recombinant IFN-γ was obtained from PromoKine (Heidelberg, Germany). TLR2 blocking antibody was acquired from eBioscience (T2.5, San Diego, USA). Anti-LPS antibody (L. pneumophila specific; ABIN235748) was obtained from antikörper-online.de (Aachen, Germany). Anti-IRAK-1 antibody was purchased from Cell Signaling (4359S), anti-tubulin antibody was from Santa Cruz (sc-5286) as well as p65 antibody (sc-372) and β-actin antibody (sc-1616). All chemicals used were of analytical grade and obtained from commercial sources.