Figure S1. RGMa, NEO1, NTN1 and TGFβ1 mRNA expression in human brain tissues and cultured neural cells. The expression of (a) RGMa, (b) NEO1, (c) NTN1, (d) TGFβ1 and (e) G3PDH mRNAs was determined by RT-PCR. The lanes represent (1) the human frontal cerebral cortex (CBR) without inclusion of the reverse transcription (RT) step, (2) CBR with inclusion of the RT step, (3) cultured astrocytes (AS), (4) cultured neuronal progenitor (NP) cells, (5) NTera2 teratocarcinoma-derived neurones (NTera2N), (6) SK-N-SH neuroblastoma, (7) IMR-32 neuroblastoma, (8) U-373MG astrocytoma, (9) HMO6 microglia and (10) peripheral blood mononuclear cells (PBMC). The 100 bp ladder marker is shown on the left.

Figure S2. The specificity of anti-RGMa antibody, anti-neogenin antibody and recombinant protein probes for tissue protein overlay. (A) The specificity of anti-RGMa antibody (AF2459) and anti-neogenin (NEO1) antibody (sc-15337) was validated by Western blot of the recombinant RGMa protein tagged with V5 and the recombinant NEO1 tagged with Xpress, both of which were expressed in HEK293 cells. The panels represent (a) RGMa, (b) V5, (c) heat shock protein 60 (HSP60), an internal control of protein loading, (d) NEO1, (e) Xpress and (f) HSP60. The lanes represent (1, 3) non-transfected cells and (2, 4) the cells transfected with the vector expressing (a–c) RGMa or (d–f) NEO1. (B) The protein extract (50 μg protein/lane) of (lanes 5, 7) NTera2N and (lanes 6, 8) U-373MG was processed for Western blot for detection of (g) RGMa, (h) NEO1 and (i) HSP60. (C) The vector expressing the Flag-tagged CTFβ of APP or Flag-tagged GFP was cotransfected with the vector expressing Myc-tagged RGMa in HEK293 cells. The cellular protein extract was processed for immunoprecipitation (IP) with (j, l) anti-Flag antibody or (k, m) anti-Myc antibody. Then, the immunoprecipitates were processed for Western blot for detection of (j, l) Myc or (k, m) Flag. The lanes indicate (9, 12) input control, (10, 13) IP with anti-Flag or anti-Myc antibody and (11, 14) IP with normal mouse or rabbit IgG. (D) The recombinant Xpress-tagged RGMa or LacZ protein was processed for Western blot for detection of (o) Xpress or (p) RGMa. The lanes indicate (15) Xpress-tagged LacZ and (16) Xpress-tagged RGMa.

nan1281_sm_figureS1.tif309KSupporting info item
nan1281_sm_figureS2.pdf613KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.