Lee J. Cells were treated with LPS (100 ng/ml) for 10 min or lauric acid (100 m) for 1 h in the presence or absence of DHA (20 m). Cells were washed with serum-free DMEM and incubated with 8 g/ml fluorescein isothiocyanate-conjugated cholera toxin B (Sigma-Aldrich) on snow for 10 min. Cells were fixed with 4% paraformaldehyde for 45 min followed by incubation with 50 mm ammonium hydroxide for 10 min and were permeabilized with 0.1% Triton X-100 for 15 min. Samples were washed Mouse monoclonal antibody to KMT3C / SMYD2. This gene encodes a protein containing a SET domain, 2 LXXLL motifs, 3 nuclear translocationsignals (NLSs), 4 plant homeodomain (PHD) finger regions, and a proline-rich region. Theencoded protein enhances androgen receptor (AR) transactivation, and this enhancement canbe increased further in the presence of other androgen receptor associated coregulators. Thisprotein may act as a nucleus-localized, basic transcriptional factor and also as a bifunctionaltranscriptional regulator. Mutations of this gene have been associated with Sotos syndrome andWeaver syndrome. One version of childhood acute myeloid leukemia is the result of a cryptictranslocation with the breakpoints occurring within nuclear receptor-binding Su-var, enhancer ofzeste, and trithorax domain protein 1 on chromosome 5 and nucleoporin, 98-kd on chromosome11. Two transcript variants encoding distinct isoforms have been identified for this gene three times with bovine serum albumin (BSA) remedy (0.5% BSA, 0.15% glycine in phosphate-buffered saline). Coverslips were clogged with 5% goat serum (Zymed Laboratories Inc., South San Francisco, CA) for 45 min followed by washing with BSA remedy. Samples were incubated for 1 h with 1/100 dilution of anti-TLR4 (H-80, Santa Cruz Biotechnology) in BSA remedy followed by a 1-h incubation with 1/500 dilution in BSA remedy of Alexa Fluor 546-conjugated F(ab)2 fragment of goat anti-rabbit IgG (Invitrogen). Coverslips were washed three times with BSA remedy and phosphate-buffered saline and mounted onto glass slides. For ROS analysis, Natural264.7 cells were incubated with 10 m 5-(and 6-)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetylester (CM-H2DCFDA) (Invitrogen) for 30 min. Cells were preincubated with 20 m DHA, 2 m diphenyleneiodonium chloride (DPI) or 10 mm and and and trichloroacetic acid-precipitated. Fractions were immunoblotted with anti-TLR4, TRIF, MyD88, or flotillin-1. and staining when the lipid rafts and TLR4 were co-localized. Staining of lipid rafts and TLR4 in resting Natural264.7 cells did not show co-localization of TLR4 with lipid rafts. TLR4 was localized in condensed formations that were distributed throughout the cytoplasm (Fig. 2staining round the plasma membrane when images were merged. When Natural264.7 cells were pretreated with DHA before LPS treatment, the co-localization of TLR4 with lipid rafts was diminished. Lauric acid produced similar effects as LPS- and lauric acid-induced co-localization of TLR4 with lipid rafts was also diminished from the pretreatment of DHA. Lauric Acid Induces, but DHA Inhibits the Homodimerization of TLR4 It is known that some TLRs function as homo- or heterodimers (31). Homodimerization of TLR4 is an initial step for receptor activation (32C34). Consequently, we identified whether lauric acid promotes the dimerization of TLR4. The dimerization assay was performed as previously explained with Ba/F3 cells stably transfected with GFP-TLR4, FLAG-TLR4, and FLAG-MD-2 constructs (26). Briefly, GFP-TLR4 was immunoprecipitated, and dimerization was determined by the co-immunoprecipitation of FLAG-TLR4. As demonstrated in Fig. 3and and and were significantly different from the ideals for the control group without DHA treatment ( 0.05). For the analysis of mRNA levels of indicated genes, Ba/F3-TLR4 cells (and were pooled from your sucrose gradient. One half of the lipid raft portion was immunoprecipitated with anti-GFP antibodies and then immunoblotted with anti-FLAG antibodies. The membranes were reprobed with anti-GFP antibodies. The other half of the samples was immunoblotted with anti-flotillin-1 antibodies to show the presence of the lipid raft marker. were immunoprecipitated and immunoblotted mainly because explained above in but were lysed and fractionated from the sucrose gradient. GFP-TLR4 was immunoprecipitated with anti-GFP antibodies from lipid raft fractions and immunoblotted with anti-FLAG antibodies. and were significantly different from the ideals for the control PPQ-102 group without nystatin treatment. For the analysis of mRNA of indicated genes, Ba/F3-TLR4 cells (and and em B /em ). Natural264.7 cells pretreated with DPI also inhibited lauric acid-induced recruitment of TLR4 to lipid rafts (Fig. 7 em C /em ). Together, these results suggest that lauric acid induces dimerization and recruitment to lipid rafts in a ROS-dependent manner as does PPQ-102 LPS. Open in a separate window Physique 7. Inhibition of NADPH oxidase suppresses LPS or lauric acid-induced TLR4 dimerization. em A /em , Ba/F3 cells were pretreated with DPI (2 m) for 1 h or NAc (10 mm) for 2 h and then stimulated PPQ-102 with LPS (100 ng/ml) or ( em B /em ) 100 m lauric acid for 30 min. Cells were lysed, and GFP-TLR4 was immunoprecipitated and immunoblotted with anti-FLAG and anti-GFP antibodies. em C /em , RAW264.7 cells were pretreated with 2 m DPI for 1 h and treated with 100 m lauric acid for 30 min. Cells were lysed, and lysates were fractionated by sucrose gradient fractionation. Lipid raft fractions ( em 1C3 /em ) were collected, trichloroacetic acid-precipitated, and immunoblotted with.