Anti tlr4

HCC cells were lysed by RIPA buffer (Thermo Fisher Scientific, USA). Protein concentrations were determined by Bradford protein assay. Equal amounts of proteins were separated on SDS‐PAGE (CA) followed by electro‐transferring on the PVDF membrane (Bio‐Rad, CA). After finishing the electro‐transfer, membranes were immersed in the blocking buffer containing 5% BSA for 2 h prior to the incubation with primary antibodies, including anti‐TLR4 (Cat: MAB6248, R&D Systems, USA), anti‐MD2 (Cat: ab24182, Abcam, USA), anti‐glypican‐3 (Cat: ab66596, Abcam, USA), anti‐Sp1 (Cat: 9389, CST, USA), anti‐STAT3 (Cat: 9139, CST, USA), anti‐phospho‐STAT3 (Cat: 9145, CST, USA), anti‐HIF‐1α (Cat: 36169, CST, USA), anti‐phospho‐p38 MAPK (Cat: 4511, CST, USA), anti‐IκB‐α (Cat: 4812, CST, USA), anti‐p65 (Cat: 8242, CST, USA) and anti‐MyD88 (Cat: 50010, CST, USA), respectively. HRP‐conjugated secondary antibodies, including anti‐rabbit IgG (7074, CST, USA) and anti‐mouse IgG (7076, CST, USA), were correspondingly used to the incubate the membranes for 2 h (Cell Signaling Technology, USA). Bands were visualized under the chemiluminescence system (Bio‐Rad, USA).

The recombinant HMGB1 with a calmodulin-binding protein (CBP) tag was provided by Kevin Tracey (The Feinstein Institute for Medical Research, Manhasset, NY, USA) (23 (link)). The CBP-tagged HMGB1 or 10 µg CBP peptide alone was incubated overnight with 50 µL HEK-Blue hTLR4 cell lysates (precleared with calmodulin beads) at 4°C with gentle shaking. The mixture of HMGB1-CBP or CBP and HEK-Blue hTLR4 cell lysates was then incubated with 30 µL drained calmodulin beads for 1 h at 4°C. After extensive washing with PBS containing 0.1% Triton X-100, proteins bound to the beads were analyzed by immunoblotting with anti-TLR4 (R&D Systems) or anti-CBP antibodies.

ATRA-NB4 cells were first treated with one of the following: LPS, Anti-CD14 (R&D, Minneapolis, MN, USA), Anti-TLR4 (R&D, Minneapolis, MN, USA), Cytochalasin D (Sigma, St. Louis, MO, USA), Bay11–7082 (InvivoGen, San Diego, CA, USA), or SP600125 (Invivogen, San Diego, CA, USA). Thereafter, ATRA-NB4 cells were washed with 5% PBS solution twice. Subsequently, the ATRA-NB4 cells were incubated with either fluorescein isothiocyanate (FITC) latex beads (Polysciences, Inc., Warrington, PA, USA, average diameter 2 m) at a ratio of 1:10 (cells: latex beads) for 1 h or PKH−26 (Sigma-Aldrich, St. Louis, MO, USA) labeled Ida-ATRA-NB4 cells (2 × 10⁶) for 30 min before determining the amount of phagocytosis using a BD FACScan [13 (link),32 (link)]. The results were expressed as (a) the percentage (%) of ATRA-NB4 cells with phagocytic activity in engulfing either latex beads or apoptotic cells and (b) a phagocytosis index that indicates a fold increase relative to the phagocytic activity of untreated ATRA-NB4 cells. Part of ATRA-NB4 cells, pre-incubated with either latex beads or labeled apoptotic cells, were examined under confocal microscopy (Fluoview; Olympus, Tokyo, Japan).