Western Blot Analysis of Cell Cycle Regulators

Viable cells in PBS buffer or cell lysates in RIPA buffer were reduced with DTT in 2x Laemmli buffer and denatured at 100°C. Western blotting was carried out as previously described [41 (link)]. Target proteins were detected using anti-α-tubulin (1:10000, T5168, Sigma‒Aldrich, St. Louis, MO, USA), anti-TRMT112 (1:500, sc-398481, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin B1 (1:500, sc-245, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin E (1:500, sc-247, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin A (1:500, sc-271682, Santa Cruz Biotechnology, Dallas, TX, USA), anti-N6AMT1 (1:1000, CQA1550, Cohesion Biosciences, London, UK), and anti-GAPDH (1:2000, sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Goat anti-rabbit-HRP (1:10000, 31460, Invitrogen, Carlsbad, CA, USA) and goat anti-mouse-HRP (1:10000, 31430, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies.