Alexa fluor 488 or alexa fluor 546 conjugated secondary antibody

THP-1 cells and HDFs were grown on coverslips at a density of 10⁴/well in 24-well plates (BD Biosciences). The cells were either pretreated or not pretreated with TH1020 (3 μM) for 1 h before the treatment with FLA-ST or FLA-AA (200 ng/mL). After that, the cells were fixed and permeabilized in chilled methanol (Samchun Chemicals, Korea) for 10 min, washed with PBS, and blocked with a 3% BSA (Biosesang, Seongnam, Gyonggi, Korea) solution in PBS for 30 min. The cells were incubated with the anti-p-p65 antibody (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA) and the anti-β-actin antibody (1:1000; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4 °C overnight. After rigorous washing, the cells were incubated with an Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. After a wash with PBS, nuclei were stained with a Hoechst 33,258 solution (5 µM; Sigma-Aldrich Corp., St. Louis, MO, USA) for 10 min. Images were captured using a fluorescence microscope (Olympus IX53; Olympus Corporation, Tokyo, Japan).

Cells were seeded and cultured on glass coverslips for 24–72 h and fixed directly to the coverslip using 4% PFA for 20 min at room temperature. Fixed cells were washed in PBS and permeabilized using 0.2% Triton X-100 at room temperature for 20 min. Fixed cells were blocked in 2.5% BSA for 1 h followed by overnight incubation at 4 °C in primary antibody diluted in a blocking solution. Primary antibody information can be found in Additional file 15: Table S8A. Primary treated cells were washed 3× for 5 min with PBS and incubated for 1 h at room temperature with Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibody (Invitrogen), diluted in a blocking solution. For proximity ligation assay (PLA), fixation and permeabilization were performed as above and PLA was performed using Duolink® PLA Technology (Sigma) as per the manufacturer’s protocol. Stained cells were washed in PBS, incubated in DAPI (Invitrogen), and diluted in PBS for 5 min. Phalloidin (Invitrogen) staining was performed as per the manufacturer’s protocol. Stained cells were mounted onto a slide using ProLong™ Gold Antifade Mountant (Invitrogen). Images were captured on Zeiss LSM800 confocal microscope or Colibri Upright LED-based microscope using the ZEN microscope software. PLA signals were quantified using ImageJ.