Western blot analysis

Protein expression was determined by Western blot analysis as previously described (41, 66). Samples were separated by SDS-PAGE. Then, proteins were transferred to a nitrocellulose membrane and blocked with Odyssey Blocking Buffer (LI-COR, Lincoln, NE) for 1 h at room temperature and probed with rabbit anti-LYPLA1 (1:1,000 v/v, abcam, Cambridge, UK), rabbit anti-LYPLA2 (1:1,000 v/v, Vanderbilt Antibody and Protein Resource Core; polyclonal rabbit anti-LYPLA2 antibody was generated by the Vanderbilt Antibody and Protein Resource Core and can be obtained through contact with corresponding author, L. J. Marnett.), rabbit anti-ERK1/2 [1:1,000 v/v, Cell Signaling Technologies (CST), Danvers, MA], rabbit anti-phospho-ERK1/2 (1:1,000 v/v, CST), rabbit anti-MEK1/2 (1:1,000 v/v, CST), rabbit anti-phospho-MEK1/2 (1:1,000 v/v, CST), or goat anti-β-actin (1:5,000 v/v, Santa Cruz Biotechnologies, Santa Cruz, CA) overnight at 4°C. Membranes were washed and incubated with IR-visible anti-rabbit or anti-goat secondary Abs (1:5,000 v/v, LI-COR). Blots were visualized using an Odyssey IR Imager.