Quantitative, solution-phase profiling of multiple transcription factors in parallel

Abstract

Transcription factors are regulatory proteins that bind to specific sites of chromosomal DNA to enact responses to intracellular and extracellular stimuli. Transcription factor signalling networks are branched and interconnected so that any single transcription factor can activate many different genes and one gene can be activated by a combination of different transcription factors. Thus, trying to characterize a cellular response to a stimulus by measuring the level of only one transcription factor potentially ignores important simultaneous events that contribute to the response. Hence, parallel measurements of transcription factors are necessary to capture the breadth of valuable information about cellular responses that would not be obtained by measuring only a single transcription factor. We have sought to develop a new, scalable, flexible, and sensitive approach to analysis of transcription factor levels that complements existing parallel approaches. Here, we describe proof-of-principle analyses of purified human transcription factors and breast cancer nuclear extracts. Our assay can successfully quantify transcription factors in parallel with ~10-fold better sensitivity than current techniques. Sensitivity of the assay can be further increased by 200-fold through the use of PCR for signal amplification.

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

“…MCF10A cells were cultured in Dulbecco's modified Eagle's media (DMEM)/F-12(1:1) (Life Technologies) with 5% horse serum, 10 g/ml insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin, 0.5 g/ml hydrocortisone, and penicillin/streptomycin (Invitrogen). MDA-MB-231 human breast cancer cell lines were obtained from the ATCC and cultured in DMEM with 10% fetal bovine serum (FBS), 2 mM glutamine, and penicillin/streptomycin (Invitrogen) as described (31,32). Cells were maintained at 37°C and 5% CO 2 environment.…”

IPAF inflammasome is involved in interleukin-1β production from astrocytes, induced by palmitate; implications for Alzheimer's Disease

“…Protein samples 15-30μg were subjected to Western analysis as previously described (Bilgin et al, 2013; Wu et al, 2013) using pro-IL-1β antibody (Biovision, Milpitas, CA, USA), caspase-1 (Abcam, Cambridge, MA, USA), IPAF (Santa Cruz biotech, Dallas, Texas, USA), ASC (Novus Biologicals, Littleton, CO, USA), CREB and pCREB (Cell signaling, Danvers, MA, USA), beta-actin and TBP (TATA binding protein) (Sigma). Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were purchased from Thermo Scientific, and donkey anti-goat IgG-HRP was purchased from Santa Cruz biotech.…”

Palmitate induces transcriptional regulation of BACE1 and presenilin by STAT3 in neurons mediated by astrocytes

“…Then the cells were lysed and protein concentrations from whole cell extracts were determined by Bradford assay (BioRad). 15-30μg of protein samples were used for Western blot analysis as previously described (Bilgin et al, 2013; Liu et al, 2013; Wu et al, 2013) using specific antibodies for BACE1 (Cat#ab2077, Abcam, Cambridge, MA, USA), pSTAT3 (Cat#9131, Cell signaling, Danvers, MA, USA), spectrin (Cat# MAB1622, Millipore, Billerica, MA, USA), Histone H1 (Cat#ab11079, Abcam, Cambridge, MA, USA), C99 (Cat#2136, QED Bioscience, San Diego, CA, USA), and beta-actin (Cat# NB600-501H, Novus Biologicals, Littleton, CO, USA). Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were purchased from Thermo Scientific (Cat#31430, 31460, Asheville, NC, USA).…”