The following reagents were used: CpG oligodeoxynucleotides (5′-TCGTCGTTTCGTCGTTTTGTCGTT-3′, Eurofins MWG Operon, Huntsville, AL), ultrapure LPS 0111:B4 (Sigma), flagellin (Invivogen), and TNF-α ELISA kit (Biolegend). The following antibodies were used: hemaglutinin tag (HA, ABM and Roche), flag tag (Sigma), tubulin (eBioscience), green fluorescent protein (GFP, life technologies), and horseradish peroxidase-labeled secondary antibodies (Southern Biotech). TLR9471-1032-HA and TLR9441-1032-HA were generated by PCR sewing using mouse TLR9-HA (Fig 1 ), and the mouse IgκB leader sequence from pDisplay (Invitrogen). Two fragments were generated with primers 1 and 2, and 3 and 4 (Table 1 ). Those products and primers 1 and 4 were included in a second PCR reaction to generate the sewed product, which was then cloned into pcDNA3.1+(see Fig. 1 for various TLR9 mutants).
Horseradish peroxidase labeled secondary antibodies
Manufactured by Southern Biotech
Horseradish peroxidase-labeled secondary antibodies are laboratory reagents used in various immunoassay techniques. They are designed to detect and amplify the signal of target biomolecules by binding to primary antibodies. The horseradish peroxidase enzyme conjugated to these secondary antibodies can catalyze a colorimetric or chemiluminescent reaction, enabling the visualization and quantification of the target analytes.
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Lab products found in correlation
2 protocols using horseradish peroxidase labeled secondary antibodies
1
TLR9 Mutant Generation and Characterization
2
Protein Extraction and Western Blot Analysis
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Tissues removed from nude mice at different post-treatment time points were hard frozen in the liquid nitrogen and stored at −80 °C. Total proteins were extracted from tissues by GentleMax tissue disruption in urea buffer (6 M Urea, 5 mM sodium fluoride, 2.5 mM sodium pyruvate, 1 mM EDTA, 0.5% Triton, 1 mM sodium orthovanadate) supplemented with protease inhibitors (Protease inhibitor cocktail tablets, Roche®).
Protein concentration determination, separation under reducing conditions and revelation were realized as described above. Immunoblotting was done with the indicated antibodies; Anti-Phospho-p53 (Ser15) Cell signaling (#9284), Anti-Phospho-Histone H2AX (Ser139) Cell Signaling (#2577), Anti-GAPDH Santa Cruz (FL-335: sc-25778) followed by corresponding horseradish peroxidase-labeled secondary antibodies (Southern Biotech). Development, imaging and relative quantification were performed as described above.
Protein concentration determination, separation under reducing conditions and revelation were realized as described above. Immunoblotting was done with the indicated antibodies; Anti-Phospho-p53 (Ser15) Cell signaling (#9284), Anti-Phospho-Histone H2AX (Ser139) Cell Signaling (#2577), Anti-GAPDH Santa Cruz (FL-335: sc-25778) followed by corresponding horseradish peroxidase-labeled secondary antibodies (Southern Biotech). Development, imaging and relative quantification were performed as described above.
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