Journal: PLOS ONE

Article Title: Epigallocatechin gallate regulates the myeloid-specific transcription factor PU.1 in macrophages

doi: 10.1371/journal.pone.0301904

Figure Lengend Snippet: A) Total cell lysates from BMDM were incubated in presence of 25 μM EGCG and 1x β-Mercapto ethanol separately and immunoblotted with PU.1 antibody to determine the monomeric or dimeric state. Relative expression levels of COX2, iNOS and β-actin were determined in BMDM pretreated with EGCG, later stimulated with LPS, and B) relative densitometry. C) BMDM nuclear protein extracts were incubated with biotin labeled TLR4 promoter oligo in presence of increasing concentrations of EGCG. The reaction mix was electrophoresed on native polyacrylamide gel in 1X TBE, transferred to Biodyne A membrane and detected by Streptavidn-HRP chemiluminescence detection kit. D) BMDM were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 2 hours. Cells were cross-linked with 1% formaldehyde and PU.1 bound TLR4 promoter was immunoprecipitated, detected by qPCR after sequential washes, de-cross linking and purification steps. PU.1 recruitment on to TLR4 promoter was expressed as % input of chromatin. Graph shows means plus SD for triplicate samples and is representative of 2 independent experiments. Data are shown as mean ± SEM. N = 5 for each group, D **** p < 0.0001 Normal IgG immunoprecipitated chromatin vs Anti-PU.1 IgG immunoprecipitated chromatin.

Article Snippet: The DNA-protein complexes on the membrane were detected by Pierce biotin-streptavidin HRP chemiluminescence detection kit (Waltham, MA).

Techniques: Incubation, Expressing, Labeling, Membrane, Immunoprecipitation, Purification