Alexa647 anti mouse secondary antibody

100 ng WT or HV SLC4A5 double-stranded DNA were generated using oligonucleotides matching either the WT G allele or variant A allele, and then annealed and incubated with 10 ng purified V5 myc epitope-tagged HNF4A protein (Origene). ChIP-grade streptavidin magnetic particles were incubated with 100 ng polyclonal mouse anti-myc antibody (Santa Cruz, clone 9E10) for 30 min and washed 3 times with PBS. They were incubated for another 30 min with Alexa647 anti mouse secondary antibody (Invitrogen), washed three times, and the fluorescence measured by microplate fluorometry (PherastarFS). The WT SLC4A5 oligonucleotides at rs7571842 were 5’ biotinylated-GTCTGTAAAACTAAGGAGGTAATTTGCTGCAACAG and the non-biotinylated complement. The variant SLC4A5 rs7571842 A allele oligonucleotides were 5’ biotinylated -GTCTGTAAAACTAAGAAGGTAATTTGCTGCAACAG and its non-biotinylated complement. The second WT oligonucleotides at rs10177833 were 5’ biotinylated-ACCCTGGCAATGTGGACACACACCCCATTCAG and the non-biotinylated complement. The 2^nd variant (A allele) oligonucleotides at SLC4A5 rs10177833 were 5’ biotinylated -ACCCTGGCAATGTGGAAACACACCCCATTCAG and the non-biotinylated complement. The positive control consensus HNF4A binding site oligonucleotide was 5’ biotinylated -AGTTCAAAGGTCA.

For TLR-4 staining, 5 × 10⁵ cells were incubated with anti-TLR4-APC (eBiosciences) for 30 min in 0.5% BSA in PBS on ice; for RAGE staining, cells were incubated with mouse anti-RAGE (Abcam) for 30 min on ice, washed with PBS and then incubated with an Alexa 647 anti-mouse secondary antibody (Invitrogen). The data were collected using a LSRII (BD Pharmingen, San Diego, CA), and the results were analyzed using Flowjo 6.3.4 software (TreeStar). After confirmation of their presence, DU145 cells were pretreated with 2.5 μg/ml anti-TLR4 (R&D Systems), 2.5 μg/ml anti-RAGE (R&D Systems), 2.5 μg/ml each of both antibodies, or control medium for 30 min in 6 well plates, before adding 1.0 μg/ml recombinant HMGB1 or supernatants harvested from DTX-treated DU145 tumor cells. After further 24 h incubation, cells were lysed and subjected to immunoblotting with anti-clusterin as described above.