Ab51841

The following antibodies were used in this study. For ChIP: Flag- M2 resin (Sigma), Trf2: in house rabbit polyclonal antibody 1727 raised against a peptide corresponding to Trf2 amino acids 115–136, gift from L. Tora, TBP: ab28175, Abcam and ab51841, Abcam, PolII: sc 9001X, Santa Cruz, H3K4Me3: 04-745, Millipore. For immunoblots the following antibodies were used. Trf2; 1727 as described above, TFIIA: rabbit polyclonal J17, gift from H.G. Stunnenberg, ALF: in house mouse monoclonal 2B11 as described35 (link), Taf7l: in house mouse monoclonal 46TA as described36 (link), TBP: ab51841 Abcam, TAF4: in house mouse monoclonal 32TA as described53 (link). TAF1: ABE42, Millipore, Actb in house mouse monoclonal 1ACT-2D7, H3: ab1791 Abcam.

Cells were grown on coverslips (Azn Scientific #ES0117650) that were pre-washed in 70% ethanol and coated with gelatin in tissue culture-treated six-well plates. After indicated treatments, cells were washed with 1 mL of PBS and fixed in 4% paraformaldehyde (UBC Chemical Stores #OR683105) for 15 min. After fixation, cells were washed and permeabilized with 1 mL 0.025% Triton-X for 5 min, followed by two 10 min washes with PBS. Samples were blocked by nutating in PBS 5% BSA for 30 min. TBP (Abcam ab51841) was diluted at 1:100 in PBS, and added to cells in coverslips for 1 hr. Samples are washed twice with PBS (5 min for each wash). Coverslips were then incubated in Alexa Fluor 594 (A11005) 1:100 for 1 hr, and washed with PBS twice (5 min for each wash). Samples are then incubated with DAPI (300 nM in PBS) for 5 min and washed twice with PBS (5 min for each wash). Coverslips were assembled using Vectashield mounting medium (BioLynx #VECTH1000). Fluorescent images were collected using the Leica DMI6000B inverted fluorescence microscope. Quantification was done through Fiji (Schindelin et al., 2012 (link)).

MCs in culture and renal medulla from rats were disrupted with lysis buffer (50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 µg/mL phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, and 1% Triton X-100). Total proteins were extracted and purified with 100 µL of chloroform and 400 µL of methanol. Protein samples were boiled for 3 min and subjected to electrophoresis on 8% polyacrylamide gels and then transblotted to nitrocellulose membranes (Bio-Rad Laboratories). Blots were incubated with rabbit polyclonal antibody for TGF-β1 (Proteintech Group, Rosemont, IL, USA) diluted 1:200, USF1 (Alexa-594 Goat anti-Rabbit IgG (A11072, Invitrogen)) diluted 1:500, rabbit anti-GAPDH antibody (ab9486, abcam) diluted 1:2000 as a cytoplasm control, and Mouse Anti-TATA binding protein (TBP, ab51841, abcam) diluted 1:2000 as a nucleus control in 5% non-fat milk in TBST solution (10 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) for 3 h at room temperature. The membrane was incubated with horseradish peroxidase-labeled secondary antibody (Sigma-Aldrich) for 1 h at room temperature and then washed with TBST once for 15 min and then more four times for 5 min. Immune complexes on the membrane were detected by the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).