Anti tlr4

Cells were seeded onto LabTek chambers (Nunc, Rochester, NY, USA) until confluence and were then stimulated for 16h in the chambers. Cells were then washed twice with pre-warmed (37°C) PBS containing Ca⁺⁺ and Mg⁺⁺ and then fixed for 30min at room temperature with PBS Ca-Mg containing 2% PFA and 2% sucrose. After fixation, we performed two quenching steps with PBS containing glycine 0.1M and NaN₃ 0.01% for 10min and a permeabilization step using PBS containing 0.2% saponin, 2% BSA, for 20min at room temperature. Staining was performed using the anti-ICAM-1 primary antibody clone 2D5 with a secondary goat anti-mouse antibody coupled to TRITC (Jackson ImmunoResearch); anti-TLR4 (Santa Cruz, Santa Cruz, CA, USA) with a secondary goat anti-rabbit antibody coupled to Alexa 488; or the anti-golgin 97 (Invitrogen) with a secondary goat anti-mouse antibody coupled with Alexa 555. Nuclei were labeled with Hoechst 33342. Cells were observed with an epi-fluorescence microscope (Carl Zeiss AxioImager) with the pseudo-confocal module APOTOME. Images were analyzed with ImageJ software (ImageJ, NIH, USA). For co-localization experiments, the Pearson’s correlation coefficient (Rr) and the Mander’s overlap coefficient (R) were calculated using the ImageJ co-localization plugin after background subtraction.

RAW264.7 cells were plated in 100-mm dishes and stimulated with LPS for 1 h. The cytoplasmic and nuclear proteins were extracted following the procedure described previously.^{47 (link), 48 (link)} Preparation of total protein extracts from mice liver was performed. The extracted proteins were separated by SDS-PAGE 12% polyacrylamide gel and then electrically transferred to a PVDF membrane. After blocking with 5% (w/v) BSA in TBST at room temperature for 1 h, the membranes were then incubated with an appropriate specific primary antibody (anti-NF-κB p65, 1 : 500; anti-β-actin, 1 : 1000; Bioworld, Minnesota, MN, USA. anti-CD14, 1 : 500; anti-TLR4, 1 : 500; Santa Cruz Biotechnology) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibody (1 : 10 000; Sunshine Biotechnology, Nanjing, China) and detected by enhanced chemical luminescence kit (Thermo Scientific, Hudson, NH, USA).