HEK293T cells were transfected with either GFP-p204 or FLAG-Casp-11 (Addgene) expressing construct using Lipofectamine® 2000 according to the manufacturer's instruction for 48 h, and pull-down assay was performed as described previously (Shi et al., 2014 (link)) with slight modification. Briefly, the transfected cells were lysed in the lysis buffer (50 mM Tric-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, proteinase inhibitor cocktail, PMSF and sodium orthovanadate) for 30 min on ice followed by centrifugation at 16,000 ×g for 10 min at 4 °C to collect supernatant. Supernatant was incubated with streptavidin agarose resin (Thermo Fisher Scientific) for its pre-clearance for 1 h at 4 °C with constant rotation. Biotin conjugated LPS (Biotin-LPS; InvivoGen) was immobilized onto streptavidin agarose resin, and unbound Biotin-LPS was removed by washing the resin three times with the lysis buffer. Pre-cleared supernatant was added to the Biotin-LPS bound streptavidin agarose resins for 1 h at 4 °C with constant rotation, and the resins were washed three times with the lysis buffer. The precipitates were eluted in 1 X SDS sample buffer followed by Western blot analysis.
Biotin lps
Manufactured by InvivoGen
Sourced in United States
Biotin-LPS is a lipopolysaccharide (LPS) molecule conjugated with biotin. LPS is a major component of the outer membrane of Gram-negative bacteria and is commonly used in research to stimulate immune responses. The biotin moiety allows for easy detection and tracking of the LPS molecule using streptavidin-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
Anti lamp1, by Abcam (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Anti rab11, by Cell Signaling Technology (1 mentions)
Anti tak1, by Cell Signaling Technology (1 mentions)
Anti rab8a, by Cell Signaling Technology (1 mentions)
Pam3csk4 biotin, by InvivoGen (1 mentions)
Anti myd88, by R&D Systems (1 mentions)
Anti rab5a, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Cba mouse inflammation kit, by BD (1 mentions)
Lps l2887, by Merck Group (1 mentions)
Mab531, by R&D Systems (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Hta125, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Horseradish peroxidase hrp, by Beyotime (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Beyotime (1 mentions)
Streptavidin agarose resin, by Thermo Fisher Scientific (1 mentions)
9 protocols using biotin lps
1
Biotin-LPS Pull-Down Assay for Protein Interactions
2
LPS Binding Assay for XN Interactions
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To determine if XN binds to MD-2, CD14 or TLR4, a LPS binding assay adapted from Zhang et al. was used [38 (link)]. In brief, polystyrene 96 well plates (R&D Systems Inc., Minneapolis, MN, USA) were coated with MD-2 (#NB100-56655; Novus Bio, Centennial, CO, USA), CD14 (#56082S, Cell Signaling Technology Inc, Danvers, MA, USA) or TLR4 (#NB100-56723SS; Novus Bio, Centennial, CO, USA) antibodies over night at 4 °C. Plates were blocked with 3% BSA for 2 h followed by an incubation with their respective recombinant protein (MD-2: 1787-MD, Bio-Techne Corp., Minneapolis, MN, USA; CD14: 110-01, PeproTech, Cranbury, NJ, USA; TLR4: 160-06, PeproTech, Cranbury, NJ, USA) (1 μg/mL) for 1.5 h. XN in concentrations ranging from 0–8 μg/mL derived through the XN-rich hop extract was added to the plate in presence or absence of LPS-biotin (50 ng/mL, InvivoGen, CA, USA,) for 1 h. After a 1 h incubation with HRP-Streptavidin (Bio-Techne Corp., Minneapolis, MN, USA) plates were incubated with TMB (Thermo Fisher Scientific, Waltham, MA, USA) for another 15 min. Excess antibody and streptavidin-HRP solution was removed by washing with 0.05% PBST. To stop the reaction, 2 N H2SO4 was added to the plate and absorbance was measured at 450 nm.
3
Bead-based TLR2 and TLR4 Activation Assay
4
Antibody Staining and Signaling Assays
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Unconjugated and Alexa 647-conjugated anti-mouse uPAR antibody were from R&D Systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from R&D Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals. Human IL-6 and IL-8 ELISAs were from Thermofisher Scientific. Mouse inflammation CBA kit was from BD Biosciences. Mouse and human uPAR siRNA and non-sence siRNA control duplexes were from Santa Cruz Biotechnology. Fluorescein isothiocyanate (FITC) was from Sigma.
5
Quantifying TLR4 Binding via Yeast Display
6
ELISA for MD-2 Binding Inhibition
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ELISA for determination of L2H21's competition against LPS for binding to MD‐2 was performed in 96‐well plates. The 96‐well plates were coated with MD‐2 antibody at 4°C overnight and blocked with 3% bovine serum albumin (BSA) for 2 hrs at room temperature. Then, rhMD‐2, rhMD‐2/R90A or rhMD‐2/Y102A protein (4 μg/ml, respectively) diluted in 10 mM Tris‐HCl (pH 7.5) solution was added to the plate, incubated for 1.5 hrs and biotin‐LPS (InvivoGen, San Diego, CA, USA) was added to the plate in the presence or absence of L2H21 (0.1 or 1.0 μM). After incubated with horseradish peroxidase (HRP; Beyotime Biotech, Nantong, China) for 1 hr at room temperature, TMB (Beyotime Biotech) was added to the plate under dark condition for 15 min. The reaction was finally stopped with 2 N H2SO4 solution. The absorbance values were measured at 450 nm.
7
LPS-Mediated Signaling Pathway Analysis
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25HC, LPS (E. coli O111:B4) and fluorescein isothiocyanate‐labelled LPS (FITC‐LPS) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant human MD‐2 (rhMD‐2) protein was obtained from R&D Systems (Minneapolis, MN, USA). Biotin‐LPS was purchased from Invivogen (San Diego, CA, USA). Anti‐MD‐2 antibody was from Abcam (Cambridge, UK). Antibodies against phospho‐Akt, phospho‐NF‐κB p65, phospho‐JNK mitogen‐activated protein kinase (MAPK), phospho‐Erk42/44 MAPK, phospho‐p38 MAPK, Akt, NF‐κB p65, MAPKs of JNK, Erk42/44, p38, and β‐actin were purchased from Cell Signaling Technology (Boston, MA, USA).
8
HMGB1 Redox State and LPS Binding Analysis
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Serum was incubated with biotin-IAM (1 μg/ml; Sigma Aldrich, St. Louis, MO, USA; B2059) for 18 h. Mixtures were incubated with 20 μl (50% slurry) of streptavidin-agarose beads (Pierce, Rockford, IL, USA; #20349) for 1 h at 4℃ with rotation and centrifuged at 8000 rpm for 1 min. The pellets contained reduced-HMGB1, while the supernatants contained disulfide-HMGB1. The supernatants were incubated with biotin-M (1 μg/ml; Sigma Aldrich, St. Louis, MO, USA; B1267) in the presence of dithiothreitol (10 mM) for 6 h and then with streptavidin beads for 1 h and then centrifuged at 8000 rpm. The pellets were washed three times and analyzed by immunoblotting. To show that HMGB1 binds to LPS, serum was preincubated with HMGB1 A box peptide (0.5 or 1 μg/ml; HMGbiotech, Milano, Italy; HM-012) for 6 h and then incubated with biotin-LPS (5 μg/ml; Invivogen, San Diego, CA, USA; tlrl-bblps) for 24 h. The mixture was precipitated using streptavidin-agarose beads for 1 h at 4℃ with rotation and analyzed by immunoblotting with anti-HMGB1 antibody (Abcam, Cambridge, UK; ab67281).
9
LPS-Induced Pull-Down Assay in 293T and BMDM Cells
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The pull-down assay was performed, as previously described (7 (link)). Briefly, 293T cells seeded in 6-well plates were transfected with the indicated plasmids using Xfect polymer according to the manufacturer's instructions. For primary BMDMs, the cells were primed with 100 ng/ml LPS for 4 h. At 48 h post-transfection or 4 h post-LPS stimulation, the cells were lysed with Triton buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-10, and protease inhibitor cocktail). After removing the cell debris, the supernatant was incubated with biotin-LPS (Invivogen, 0111:B4) and NeutrAvidin beads (Thermo Fisher Scientific, 29201) overnight at 4 °C. Then, the beads were washed three times with the Triton buffer, after which the precipitates were eluted with 2 SDS loading buffer and boiled for 5 min at 95 °C.
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