Hrp substrate

Protein samples were separated on an SDS-PAGE gel, followed by transfer to nitrocellulose membrane (GE Healthcare Lifesciences) using the Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories). After 1 h incubation with 1% (w/v) nonfat milk in 1X TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) at room temperature, membranes were incubated with primary antibodies overnight at 4°C. After three washes with 1X TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), HRP-conjugated secondary antibodies were added. Membranes were then washed three times with 1X TBST and incubated with HRP substrates (Li-COR Biosciences, Lincoln, NE). The signals were detected with ChemiDoc and quantified using ImageJ. Graphing and statistical analyses were performed using the built-in functions of Prism. The raw quantification data used for graphing were listed in S7 Dataset.
For immunoblotting, polyclonal antibodies against TFIIIA were diluted as 1:2,000 and the monoclonal 8WG16 antibodies (Thermo Fisher Scientific) were diluted at 1:1,000. HRP-conjugated anti-mouse serum (Bio-Rad) was diluted at 1:5,000. HRP-conjugated anti-rabbit serum (Thermo Fisher Scientific) was diluted at 1:3,000. For silver staining, we followed the instructions of the Silver BULLit kit (Amresco, Solon, OH).

The purified protein samples together with the BLUItra prestained protein ladder (FroggaBio, Wheatfield, NY, USA) were separated on an SDS-PAGE gel, followed by transferring to an Amersham Protran 0.45 NC nitrocellulose membrane (GE Healthcare Lifesciences, Pittsburgh, PA, USA) using the Mini-PROTEAN Tetra Cell (BioRad, Hercules, CA, USA). After 1 h incubation with 1% (w/v) nonfat milk in 1X TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) at room temperature, the 8WG16 monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA) at 1:1000 against the largest subunit of Pol II was added and incubated overnight at 4 °C. After three washes with 1X TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), HRP-conjugated secondary antibody against mouse IgG (Millipore Sigma, Burlington, MA, USA) was added at 1:8000 dilution. Membrane was washed three times with 1X TBST and incubated with HRP substrates (Li-COR Biosciences, Lincoln, NE, USA). The signals were detected with C-DiGit (Li-COR Biosciences, Lincoln, NE, USA).