Sc 421

Proteins separated by SDS-PAGE, were transferred onto PVDF membranes and immunoblotted with antibodies anti-double phosphorylated ERK1/2 (dilution 1:5000) (#9101, Cell Signalling Technologies, Danvers, MA, USA) and anti-phosphoTyr ERK (dilution 1:1000) (#sc-7383, Santa Cruz Biotechnology Inc., Dallas, TX, USA). After cell transfection, anti-V5 tag (dilution 1:1000) (#ab 27671, SV5-Pk1, Abcam, Cambridge, UK) was used to detect specifically recombinant over-expressed ERK2 protein. Anti-complex III or TOM40 (dilution 1:5000) (#ab 14745, Abcam, Cambridge, UK) and anti-polimerase II (POLII) or anti-TFIID (or TBP) (#ab sc-421, Santa Cruz Biotechnology Inc., Dallas, TX, USA) (dilution 1:1000) were used as mitochondrial and nuclear loading control respectively, and antibodies against actin (dilution 1:4000) were used for cytosolic fraction characterization (Upstate, Fisher Scientific, Thermo Fischer Scientific, #05661). Cells were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (dilution 1:5000; #1706515 and #1721011, respectively; Bio-Rad Laboratories Inc.). Chemiluminescence was developed with enhanced ECL reagent (GE Healthcare, Buckinghamshire, UK) and bands were detected by autoradiography (X100 Autoradiography film, GE Healthcare). Acrylamide and PVDF membranes were from Bio-Rad Laboratories Inc.

HepG2, HuH-7, and 293T cells were cultured in 6-well plates. At 90% confluence, cells were harvested and lyzed with 1×SDS loading buffer. After heating at 100°C for 10 min, 20 μL cell lysate was loaded for SDS-PAGE electrophoresis and western blot analysis. Western blot was performed using antibodies against STAT3 (CST#9139, CST), mCherry (TAG0080, Frdbio), GAPDH (RAB0101, Frdbrio), BRF1 (SC-81405, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), GTF3C3 (SC-393235, Santa Cruz Biotech), TBP (SC-421, Santa Cruz Biotech), and TP73 (CSB-PA003696, CUSABio).