Four × 106 BMDM per well were plated in 6-well plates and treated with CPDP for 30 min prior to stimulation with ODN 1668 for 90 min. Cells were lysed in 600 μL of buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM NaF, 2 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1 mM DTT, and protease inhibitor cocktail (Roche, Indianapolis, IN). Cell extracts were incubated overnight with 1 μg of anti-mouse MyD88 antibody (AF3109) (R&D Systems; Minneapolis, MN), followed by 4 h incubation with 20 μl protein G Sepharose beads (Thermo Fisher Scientific, Waltham, MA). The beads were then washed 3 times with 500 μL of lysis buffer and boiled in 60 μL of Laemmli sample buffer (Bio-Rad; Hercules, CA). Cell extracts were electrophoresed on 10% acrylamide gel by SDS-PAGE and transferred to PVDF membrane (Bio-Rad; Hercules, CA). Rabbit anti-MyD88 IgG was purchased from Cell Signaling (Danvers, MA). Mouse anti-IRAK4 IgG was obtained from Abcam (Cambridge, United Kingdom). Alkaline phosphatase-conjugated secondary antibodies against mouse and rabbit IgG were purchased from Cell Signaling (Danvers, MA). The stabilized substrate for alkaline phosphatase was from Promega (Madison, WI).
Stabilized substrate for alkaline phosphatase
Manufactured by Promega
Sourced in United States
Stabilized substrate for alkaline phosphatase. A ready-to-use solution that provides a stable and consistent substrate for the detection and quantification of alkaline phosphatase activity in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Protein g sepharose beads, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Intermediate ripa lysis buffer, by Beyotime (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
2 protocols using stabilized substrate for alkaline phosphatase
1
MyD88-IRAK4 Signaling Pathway Activation
2
Western Blot Analysis of ER Stress Markers
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Cells were gently washed three times in 1× PBS and then 50 μL of intermediate RIPA Lysis Buffer (Beyotime) was added per 24-well plate. Protein concentrations were detected using bicinchoninic acid (BCA) protein assay kits (Cat. No. P0010; Beyotime). Protein samples (approximately 80 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The primary antibodies used were as follows: Phospho-PERK (3179S, CST, dilution of 1:1000), PERK (3192, CST, dilution of 1:1000), Phospho-eIF2α (9721, CST, dilution of 1:1000), eIF2α (9722, CST, dilution of 1:1000), Caspase-3 (9662, CST, dilution of 1:1000), Parp-1 (9532, CST, dilution of 1:1000), Bcl-2 (4223, CST, dilution of 1:1000), ATF-6 (65880, CST, dilution of 1:1000), IRE1α (3294, CST, dilution of 1:1000), and β-actin (sc-130657, Santa Cruz, dilution of 1:800). The secondary antibody was anti-rabbit (s3738, Promega, dilution of 1:7500) alkaline phosphatase-conjugated antibody. Protein coloration was performed using the Stabilized Substrate for Alkaline Phosphatase (S3841, Promega) and was screened by the FluorChem R system (ProteinSimple, San Jose, CA, USA).
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