Figure 1 (See previous page).

N-terminal of Nogo-(B)is cleaved in Ras-transformed cells. (A) Comparison of cell surface proteins between non-transformed and Ras-transformed NIH-3T3 cells. Cell surface proteins were labeled with membrane impermeable biotin, then isolated using streptavidin-conjugated magnetic beads. Isolated proteins were analyzed by SDS-PAGE/silver nitrate staining. Bands showing different intensity levels were excised and identified by mass spectrometry. Arrows indicate proteins containing Nogo protein peptides. The 43 kDa band(s) represents Nogo-B, and the lower band (˜34 kDa) likely represents its cleavage product. (B) Schematic diagram of Nogo isoforms. Three major isoforms of Nogo (A, B, and C) share C-terminal region (shaded in black). Nogo-A and -B share N-terminal region of 167 a.a. (light gray). Peptide sequences detected by mass spectrometry were shown in Nogo-B structure. Antibodies used for detecting C-terminal and N-terminal ends of Nogo were indicated ('Y' in black and for C-terminus and 'Y' in light gray for N-terminus). (C) Detection of Nogo-B in total cell lysates (left panel) and membrane fractions (right panel) from non- and Ras-transformed cells by western blot using the antibody detecting the C-terminus of Nogo-B. β-actin and Calnexin (ER membrane marker) were used as loading controls. (D) Cell lysate of non- or Ras-transformed cells were fractionated by differential centrifugation (Lys, lysate; Nuc, nucleus; Mit, mitochondrial fraction; Cyt, cytosolic fraction; Mic, microsomal fraction). Western blots were carried out using the anti-C-terminal antibody (upper panel), or the anti-N-terminal antibody (lower panel). Cox IV, Calnexin, and β-actin were used as mitochondrial, ER, and cytosol markers, respectively. (E) mRNA levels of Nogo-B in non-transformed and Ras-transformed cells were compared using RT-PCR with Nogo-B specific primers. GAPDH was used as a loading control. (F) Ras-transformed cells were treated with vehicle control (DMSO), caspase inhibitor (Z-VAD-FMK, 10 μM), proteasome inhibitor (MG-132, 1 μM) or both for 18 hrs. Full-length Nogo-B is indicated as “FL” and a cleavage product as “C.”