Rna polymerase 2

Phosphospecific JNK, AKT, p38, p65, IκBα and ERK antibodies and TNF-α-neutralising antibody were all purchased from Cell Signaling Technology. NORE1A monoclonal antibody, CD40L, p21, p53, c-Rel, RNA polymerase II, rabbit isotype and histone H3 antibodies were all from Santa Cruz Biotechnology. CD40L–APC and isotype–APC conjugates were from eBioscience. For immunoblotting, 10–50 mg protein was separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane (for phosphoproteins) or Biotrace nitrocellulose membranes (Pall, Port Washington, NY, USA) (for nonphosphoproteins), and blocked with 10% of BSA (for phosphoproteins) or nonfat milk (for nonphosphoproteins) dissolved in PBS supplemented with 0.1% Tween 20 for 1 h. After three washes with PBS supplemented with 0.1% Tween 20, membranes were incubated overnight at 4 °C with primary antibody and for 1 h at room temperature with the appropriate secondary antibody followed by ECL (Amersham Biosciences, Piscataway, NJ, USA). Cytoplasmic and nuclear extracts from EJ cell were prepared by using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, MA, USA, cat. 78835) according to the manufacturer's instructions.

H4IIE cells were maintained in DMEM (Invitrogen) and harvested untreated or 3 and 7 h following rhBMP6 (100 ng/mL) application. The standard ChIP protocol was performed as described [12 (link)], except of using antibodies against Smad1,2,3,5,8 (Santa Cruz Biotechnology), RNA polymerase II (Santa Cruz Biotechnology) and histone H3 acetylated at lysines 9 and 14 (Diagenode). The original immunoprecipitated and total input DNA aliquots were used to assess the quality of the ChIP. The Smad occupancy was detected by qPCR on a Roche LightCycler at the insulin and PepCK promoters. The quality of the ChIP was calculated as fold enrichment over myoglobin exon 2 non-binding control region.

Ash2l (Bethyl, A300-489A), Cfp1 (Abcam ab198977), Dpy30 (Bethyl A304-296A), H3K4me1 (Abcam, ab8895), H3K4me2 (Abcam, ab7766), H3K4me3 (Abcam, ab8580), H3K9me2 (Abcam, ab1220), H3K79me2 (Abcam, ab3594), IgG (Millipore 12-370), Mll1 (Bethyl A300-086A), Mll2 (Santa Cruz, H300, sc-292359), Pax6 (Millipore, ab2237), Plekha1 (Novus, NBP1-86967), Rbbp5 (Bethyl, A300-109A), RNA polymerase II (Santa Cruz, N-20), Set1a (Bethyl, A300-289A), Snf2h (Active Motif, 39543), Spt16 (Biolegend, 607001), Ssrp (Biolegend 609702), TBP (Abcam, ab51841), vinculin (Abcam, ab129002), and Wdr5 (Bethyl A302-429A) were used.