Decreased expression of p50 or C/EBP�� was achieved by transfecting the cells with prevalidated siRNAs (Ambion). selleck chemical Volasertib The cells were transfected with the siRNA using siPORT (Ambion) 24 h prior to transfection with the reporter constructs. Luciferase detection was performed 24 h after reporter construct transfection. Expression was calculated as the relative let-7i driven Firefly luciferase to transfection control Renilla luciferase and presented as fold-change compared with empty vector pGL4.22 Firefly luciferase to Renilla luciferase, which was arbitrarily set to one. Chromatin Immunoprecipitation ChIP was performed following the protocol from Upstate. Briefly, H69 cells were cultured in the presence or absence of C. parvum sporozoites or LPS.

Chromatin was cross-linked with 1% formaldehyde, cells were lysed, and cell extract sonicated to shear DNA. Sonicated cell extract was either aliquoted as genomic input DNA or immunoprecipitated using p50 or p65 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoprecipitates containing protein-DNA complexes were washed and heated at 65 ��C for 4 h to reverse cross-link and free DNA. DNA was purified using spin columns. Semi-quantitative and quantitative PCR were performed on both genomic input and ChIP DNA. PCR primers were designed to flank the two NF��B consensus sequences in the let-7i promoter (supplemental Table S1). Quantitative PCR of the ChIP products and genomic input DNA was performed by real-time PCR using SYBR green (Roche). The amount of ChIP DNA present in each sample was reported as percentage of genomic input DNA.

Electrophoretic Mobility Shift Assay EMSA was performed following the manufacturer’s instruction (Pierce). Briefly, nuclear proteins were extracted using NE-PER (Pierce) from uninfected, C. parvum-infected, or LPS-treated H69 cells. Complementary oligonucleotides, corresponding to the NF��B consensus sequences in the let-7i promoter, were synthesized (Mayo Clinic Molecular Core Facility), annealed, and biotinylated according to the manufacturer’s instruction (Pierce). The nuclear extract was incubated for 20 min at room temperature with the biotinylated oligonucleotides. Unlabeled NF��B consensus oligonucleotides were used as competimers. These competimers were preincubated with the nuclear extract for 5 min prior to the addition of biotinylated oligonucleotides.

EMSA supershift was performed using the p50 antibody sc114x (Santa Cruz Biotechnology, Inc.). The antibodies were preincubated with the nuclear extract for GSK-3 20 min at room temperature prior to adding the biotinylated oligonucleotides. The DNA-protein complex was resolved on a 5% PAGE gel in 0.5�� TBE, transferred to a nylon membrane, and cross-linked at 120 mJ/cm2 for 60 s. The biotinylated-labeled oligonucleotides were then detected by chemiluminescence according to the manufacturer’s directions.