Fig. 1

ARMS2 expression in human monocytes and microglia cells. a Transcription of ARMS2 in uninduced or PMA (400 ng, 24 h) induced THP-1 monocytes, mouse RAW 264.7 cells, microglial cells stimulated with LPS (5 ng/ml, 24 h) or blood-derived human monocytes. b New polyclonal antiserum generated to recombinant ARMS2 detects the C-terminal part of ARMS2 as indicated by the dark spots. Peptides of ARMS2 (33 amino acids with an overlap of 3 amino acids) were spotted to a membrane and incubated with the ARMS2 antiserum. c Recombinant ARMS2 expressed in P. pastoris and purified by Ni²⁺ chromatography appears as monomeric protein of about ~15/17 kDa identified by silver staining or Western blot analysis using either monoclonal penta-Histidin, purchased polyclonal ARMS2 antiserum (αARMS2_Com) or polyclonal ARMS2 antiserum (αARMS2_Jena), generated to the recombinant protein) as indicated. The ARMS2 protein without purification tag showed a mobility of about 11 kDa. Mass spectrometry of the deglycosylated 17 kDa ARMS2 band revealed ARMS2 peptides (raw data). MS of the whole protein showed a protein with a mass of 11.349 kDa. d ARMS2 is present in monocytes with one or two copies of the non-risk ARMS2 variant, but is absent in cells with the homozygous rs10490924 (II/II) or rs2736911 (III/III) polymorphism. ARMS2 is detected in the cytoplasm of THP-1 cells (I/II), but ARMS2 is absent in RAW264.7. cells. Cells in (d) were permeabilized, stained for ARMS2 (red) or DNA (blue) and visualized by laser scanning microscopy. Scale bar = 10 μm. e Blood-derived monocytes express ARMS2 upon oxidative stress. Cells were incubated for 1 h in medium with H₂O₂ (0.001–1 mM), for another 20 h in normal medium, lysed in loading buffer, and proteins were separated by SDSPAGE and immunoblotted using αARMS2_Jena. Densitometric analysis is shown in the graph below. The same blot was also stained for β-actin as a loading control. f Immunoprecipitation of ARMS2 from THP-1 cell lysate (1 × 10⁷) with new monoclonal ARMS2 antibodies. Eluted proteins were separated by SDS-PAGE, stained with coomassie blue and single bands (white bars) investigated by mass spectrometry. ARMS2 peptides from five immunoprecipitations are shown. Precipitation of recombinant ARMS2 revealed ARMS2 petptides (data not shown). Experiments shown in a to e are representatives of three independent experiments. ∆m/z [ppm] mass tolerance, ARMS2-IP imunoprecipitation of recombinant ARMS2, THP1-IP ARMS2 immunoprecipitated from THP-1 cells