anti eaf2  (Bethyl)

Journal: Cell reports

Article Title: Multivalent role of human TFIID in recruiting elongation components at the promoter proximal region for transcriptional control

doi: 10.1016/j.celrep.2019.01.012

Figure Lengend Snippet: Key Resources Table

Article Snippet: EAF2 , Bethyl Lab , Cat# A302-503A.

Techniques: FLAG-tag, Virus, Recombinant, SYBR Green Assay, Purification, Construct, RNA HS Assay, Isolation, DNA HS Assay, Expressing, Knockdown, Western Blot, RNA Expression, Control, shRNA, Plasmid Preparation, Cloning, Software, Magnetic Beads, Gel Extraction

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.

doi: 10.1038/ncomms6941

Figure Lengend Snippet: Figure 9 | Depletion of TAF7 in cells increases the occupancy of LEC at Pol II-transcribed snRNA genes. (a) Western blot showing TAF7 and Hsp90 at 48 h after transient transfection of non-targeting (control) siRNA pool or siRNA pool targeting TAF7. (b) Effect of TAF7 depletion on transcript levels of Pol II-transcribed snRNA genes detected by RT–qPCR performed with total RNA from cells transfected with siRNAs. Ct values were normalized to GAPDH. Data points are the average of three independent experiments, and error bars show s.d. The P values for the indicated comparisons were determined by Student’s t-test (*Po0.05; **Po0.01). (c) Schematic representation of immobilized oligo(dC)-tailed template assay. Templates immobilized on streptavidin beads were incubated with purified recombinant protein EAF1 in the presence or absence of Mediator and/or TAF7 N243. After washing, bead-bound proteins were detected by western blot. (d) TAF7 interferes with EAF1 recruitment by Mediator in vitro. Upper panel shows immobilized oligo(dC)-tailed template assays. Assays were performed with an oligo(dC)-tailed template, EAF1, and Mediator in the presence or absence of TAF7 N243. Lower panel shows silver staining of Mediator and HA-tagged TAF7 N243 used in the assay. Mediator ( 1) and HA-TAF7 N243 ( 1) contain 1 pmol of the each protein or protein complex. (e) Model of immobilized oligo(dC)-tailed template assay. TAF7 N243 inhibits EAF1 recruitment to the oligo(dC)-tailed template by MED26-containing Mediator. (f) Depletion of TAF7 increased the occupancy of ICE1 (KIAA0947), EAF2 and total Pol II at U4-1 and U5B snRNA genes. Ct values of each ChIP were normalized to that of input. Data points are the average of three independent experiments, and error bars show s.d. The P values for the indicated comparisons were determined by Student’s t-test (*Po0.05; **Po0.01).

Article Snippet: Anti-FLAG M2 antibodies (1:2,000 dilutions, Sigma-Aldrich Corp.), anti-HA antibodies (1:2,000 dilutions, Covance, Princeton, NJ), anti-Myc antibodies (1:1,000 dilutions, Covance), anti-TAF7 antibodies (1:200 dilutions, sc-101167, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ELL antibodies (1:1,000 dilutions, A301-645A, Bethyl Laboratories, Montgomery, TX), anti-EAF2 antibodies (1:1,000 dilutions, A302-502A-1, Bethyl Laboratories), anti-Rpb1 antibodies (1:2,000 dilutions, sc-899 X, Santa Cruz Biotechnology) and anti-MED1 antibodies (1:2,000 dilutions, sc-5334 X, Santa Cruz Biotechnology) were used in western blots.

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Incubation, Recombinant, In Vitro, Silver Staining