Anti annexin a1

LPS derived from Escherichia coli, serotype EH100 (Enzo Life Sciences, ALX-581-010), R848 (Sigma-Aldrich, SML0196), poly(I:C) (InvivoGen, tlrl-ic), Pam3CSK4 (InvivoGen, tlrl-pms), DMF (Sigma-Aldrich, 24226), diethyl maleate (DEM; Sigma-Aldrich, D97703), and fumarate hydratase-IN-1 (MedChemExpress, HY-100004) were used. 4-OI was initially supplied by Prof. Richard Hartley, but results were confirmed and later experiments conducted using commercially available 4-OI (Sigma-Aldrich, SML2338). Abs used were anti-annexin A1 (Cell Signaling Technology, 3299), anti-NRF2 (Cell Signaling Technology, 12721S), anti-KEAP1 (Cell Signaling Technology, 8047S), anti-ABCA1 (Cell Signaling Technology, 96292), anti–IL-1β (R&D Systems, AF401-NA), and anti–β-actin (Sigma-Aldrich, AC-74). Anti-mouse IgG and anti-rabbit IgG secondary HRP-conjugated Abs (Jackson ImmunoResearch) were also used. The Silencer Select small interfering RNAs (siRNAs) against NRF2 (s70522), KEAP1 (s78526), ABCA1 (s61785), and FH (s66045) in addition to the Silencer Select negative control (Thermo Fisher Scientific) were used.

Western blotting was performed as previously described [29 (link)]. Protein samples were separated by SDS–PAGE electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes with proteins were incubated with 5% nonfat milk, specific primary antibodies at 4 °C overnight, and secondary antibodies (1:5000, BA1050, BA1054, Boster, Wuhan, China) in sequence and detected using ECL reagent. The primary antibodies were as follows: anti-Annexin A1 (1:1000, 32,934, Cell Signaling Technology), anti-E-cadherin (1:1000, ab231303; Abcam, Cambridge, U.K.), anti-vimentin (1:1000, ab20346; Abcam), anti-MMP9 (1:1000, ab76003; Abcam), anti-EGFR (1:1000, 66,455–1-Ig; Proteintech), anti-phospho-EGFR (1:1000, 3777, Cell Signaling Technology), anti-AKT(1:1000, 4685, Cell Signaling Technology), anti-phospho-AKT (1:1000, 4060, Cell Signaling Technology), anti-ERK (1:1000, 4659, Cell Signaling Technology), anti-phospho-ERK (1:1000, 4370, Cell Signaling Technology), anti-STAT3, anti-phospho-STAT3, anti-GAPDH (1:10,000, 60004–1-Ig; Proteintech), and anti-alpha tubulin(1:10,000, 66031-1-Ig; Proteintech) antibodies.

LPS derived from Escherichia coli, serotype EH100 (Enzo Life Sciences, ALX-581-010), R848 (Sigma-Aldrich, SML0196), poly(I:C) (InvivoGen, tlrl-ic), Pam3CSK4 (InvivoGen, tlrl-pms), DMF (Sigma-Aldrich, 24226), diethyl maleate (DEM; Sigma-Aldrich, D97703), and fumarate hydratase-IN-1 (MedChemExpress, HY-100004) were used. 4-OI was initially supplied by Prof. Richard Hartley, but results were confirmed and later experiments conducted using commercially available 4-OI (Sigma-Aldrich, SML2338). Abs used were anti-annexin A1 (Cell Signaling Technology, 3299), anti-NRF2 (Cell Signaling Technology, 12721S), anti-KEAP1 (Cell Signaling Technology, 8047S), anti-ABCA1 (Cell Signaling Technology, 96292), anti–IL-1β (R&D Systems, AF401-NA), and anti–β-actin (Sigma-Aldrich, AC-74). Anti-mouse IgG and anti-rabbit IgG secondary HRP-conjugated Abs (Jackson ImmunoResearch) were also used. The Silencer Select small interfering RNAs (siRNAs) against NRF2 (s70522), KEAP1 (s78526), ABCA1 (s61785), and FH (s66045) in addition to the Silencer Select negative control (Thermo Fisher Scientific) were used.