Fig. 6

GAREM2 may work as a regulator of neurite outgrowth in neurons. a The primary cultured hippocampal neurons (DIV 3) were immunostained with an anti-Tau antibody. The representative images for WT (left panel) and GAREM2 KO neurons (right panel). b Graph showing the length of total neurites for each genotype. The total neurite consists of all axons and all dendrites. Error bars indicate SEM. Scale bar, 50 μm. c Expression of FLAG-GAREM2 induced morphological alteration of SH-SY5Y cells. Images show representative 40–50 cells of the SH-SY5Y cell line. Images were taken at 48 h of the last passage of the cells in the culture dish containing normal growth medium. Images of phase contrast of control SH-SY5Y (1: left) and the A431 clone stably expressing FLAG-GAREM2 (2: right) are shown. Scale bar, 50 μm. d Association between GAREM2 and both ITSN1 and ITSN2 in SH-SY5Y cells. Immunoprecipitation (IP) experiments were carried out with total cell lysates (TCL) of the SH-SY5Y cells lysates using anti-FLAG antibody. Immunoblot (IB) analysis was carried out with anti-ITSN2 (top), anti-ITSN1 (middle), and anti-FLAG (bottom) antibodies. e Subcellular localization of FLAG–GAREM2 (red) and GFP–ITSN2 (green). COS-7 cells expressing FLAG–GAREM2 (right), GFP–ITSN2 (left). f COS-7 cells co-expressing both FLAG–GAREM2 (middle) and GFP–ITSN2 (left). FLAG-GAREM2 proteins (red) were processed for immunofluorescence staining using anti-FLAG antibody. The merged image is shown on the right. Scale bar, 10 μm