Cellular nf κb p65 translocation kit

The cells were immunofluorescence-labelled according to the manufacturer’s instruction using a Cellular NF-κB p65 Translocation Kit (Beyotime Biotech, Nantong, China). P65 protein and nuclei fluorescence as red and blue, respectively, and can be simultaneously viewed by fluorescence microscope (200x, Nikon, Tokyo, Japan) at an excitation wavelength of 350 nm for DAPI (4′,6-diamidino-2-phenylindole) and 540 nm for cyanine 3 (Cy3).

Chemical reagents, Pam3CK and lipopolysaccharide (LPS) were purchased from Sigma (Sigma, St. Louis, MO, USA). RPMI-1640, DMEM medium and FBS were obtained from Gibco (Gibco, Eggenstein, Germany). EBioscience (eBioScience, San Diego, CA, USA) was the source of mouse IL-6 ELISA Kit, mouse TNF-α ELISA Kit and anti-rhMD2 antibody. Trizol-reagent was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA). Chemiluminescent EMSA Kit, ECL detection reagent, protein A + G agarose, biotin-labeled NF-κB probe and Cellular NF-κB p65 Translocation Kit were obtained from Beyotime Biotechnology (Beyotime Biotech, Nantong, China). Anti-GAPDH, anti-IκBα, anti-p-ERK, anti-ERK, anti-CD68, anti-TLR4 and anti-MD2 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-P38, anti-P38, anti-p-JNK and anti-JNK were from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA). Recombinant human MD2 (rhMD2) protein, Recombinant human TLR4 (rhTLR4) protein, and TLR2 inhibitor CU-CPT22 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Mutated rhMD2 was obtained using the methods described in our previous publication.35 (link)