Ab109114

Cells were plated in 96-well black/clear bottom plates (Thermo Fisher Scientific, Waltham, MA, United States) and incubated overnight at 37°C and 5% CO₂. Then, cells were treated with either Sirtuin or HDAC inhibitors or siRNA. After the appropriate incubation time, NAT1 protein expression was evaluated using an in-cell western (ICW) protocol as previously described (Stepp et al., 2019 (link); Salazar-Gonzalez et al., 2020 (link)). Briefly, the cells were fixed using 3.7% paraformaldehyde and permeabilized on the plate using TBS +0.1% Triton X-100. Then, plates were blocked using 1 × fish gelatin (Biotium Inc., Fremont, CA, United States). Staining was done using rabbit monoclonal anti-NAT1 antibody (ab109114, Abcam, Cambridge, United Kingdom) and mouse monoclonal anti-β-actin, as endogenous control (A2228, Sigma-Aldrich, St. Louis, MO, United States) overnight in the cold room. The next day, plates were washed with TBS +0.1% Tween 20 (TBST) and incubated with secondary antibodies IRDye^® 800 CW goat anti-rabbit and 680RD goat anti-mouse (LI-COR Biosciences, Lincoln, NE, United States) at room temperature for 1 hour. Finally, plates were washed with TBST and scanned using an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, United States). NAT1 protein expression was normalized to β-actin content in each well.

Seven different antibodies were investigated for their specificity for NAT1 or NAT2. Two antibodies, anti-NAT1 rabbit polyclonal and anti-NAT2 rabbit polyclonal were custom designed and obtained from BioSource International (now part of Thermo Fisher Scientific, Waltham, MA, USA), referred to as DWH-NAT1 and DWH-NAT2, respectively. DWH-NAT1 immunogen sequence is CLHSDLLEDSKYR. DWH-NAT2 immunogen sequence is FLNSHLLPKKKHQ^{50 (link),54 (link)}. Briefly, the corresponding sequences for each of these antibodies were conjugated to KLH prior immunization. Then, following 1 immunization plus 4 boosts in 2 different animals; the serum was obtained and then purified using affinity columns. Other antibodies investigated were NAT1 rabbit polyclonal antiserum ES-195 (kindly provided by Professor Edith Sim, University of Oxford, UK), NAT1/2-G5 mouse monoclonal sc-137204 and NAT2 (4-KK21) mouse monoclonal sc-134399 (Santa Cruz Biotechnology, Dallas, TX, USA), recombinant anti-NAT1 rabbit monoclonal antibody [EPR3221(2)] (ab109114), and recombinant anti-NAT2/AT-2 rabbit monoclonal antibody [EPR15856] (ab194114) (Abcam, Cambridge, UK).