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Figures
- Fig. 1.
Characterization of anti-ALK monoclonal antibody. (A) Preparation of immunogen (APmH). The entire extracellular domain (ECD) of mouse ALK (ALK) followed by a PreScission protease site and a myc-His tag (PmH) was expressed and collected as described in Materials and Methods. The ECD of ALK fused with the Fc portion of human immunoglobulin (ALK-Fc) was used as an antigen for ELISA screening. Abbreviations: SS, signal sequence; TM, transmembrane domain; PTK, protein tyrosine kinase domain. (B) HEK 293T cells were transfected with APmH-expressing vector (+) and the recombinant protein was collected on the chelating Sepharose, separated by SDS-PAGE, and transferred to PVDF membranes for immunoblotting. Supernatant from cells transfected with empty vector was treated in the same way as a negative control (–). The membranes were blotted with either anti-myc (myc) or anti-ALK monoclonal (mAb16-39) antibodies (2 μg/ml). (C) APmH protein was immunoprecipitated with anti-myc antibody (myc), mAb16-39 (16-39), or normal rat IgG (IgG) (2 μg each). As a control, the APmH protein was collected by the chelating Sepharose (HIS), which allowing the purification of the recombinant protein through binding to the His tag epitope. Immunoprecipitated proteins were probed with anti-myc antibody 9E10 (myc).
- Fig. 2.
Monoclonal anti-ALK antibody 16-39 induces tyrosine phosphorylation of ALK. Untransfected NIH 3T3 cells (NIH), derivative cells expressing mouse ALK (2-C4), or human neuroblastoma cells SK-N-SH (SK) were treated with 20 nM normal rat IgG (–) or mAb16-39 (+) for 5 minutes at 37°C. Lysates were immunoprecipitated with polyclonal anti-ALK antibodies that recognize the carboxyl terminus of ALK. The phosphotyrosine level was examined by western blotting (Blot) with anti-PY antibody RC20-HRP (upper panels). The presence of equal amounts of ALK was confirmed by reprobing with polyclonal anti-ALK antibodies (lower panels). Arrowheads indicate the positions of endogenous and transfected ALK (220, 140, and 85 kDa in 2-C4 cells and 220 and 140 kDa in SK-N-SH cells). The bracket shows the position of phosphoproteins of 66, 52, and 46 kDa in both 2-C4 and SK-N-SH cells. Asterisks indicate phosphoproteins of 160, 120, 85, and 35 kDa in SK-N-SH cells.
- Fig. 3.
Dose-response (A) and time course (B) of ALK tyrosine phosphorylation in 2-C4 cells. For dose-response analysis, 2-C4 cells were treated with increasing concentrations of mAb16-39 (0, 0.12, 0.4, 1.2, 4, 12, 40, and 120 nM) for 5 minutes at 37°C. As a contr ol, parental NIH 3T3 cells were treated with 12 nM mAb for 5 minutes. For time-course analysis, cells were incubated with 4 nM mAb16-39 for the periods indicated (0, 2, 5, 15, 60, and 180 minutes and 24 hours). As controls, 2-C4 or parental NIH 3T3 cells were treated with 4 nM control IgG for 24 hours or 4 nM mAb for 5 minutes, respectively. ALK immunoprecipitates were probed with anti-PY antibody RC20-HRP (PY) and then reprobed with anti-ALK antibodies (ALK). The positions of 220-, 140-, and 85-kDa ALK bands are indicated by arrowheads.
- Fig. 4.
Analysis of downstream signaling. (A) Serum-starved SK-N-SH cells were stimulated with 4 nM anti-ALK mAb16-39 for 5 minutes. Lysates were immunoprecipitated (IP) with anti-ALK (ALK) or anti-Shc (Shc) antibodies or were immunoprecipitated without antibodies (mock) and immunoblotted with anti-PY antibody (upper panels). The same blots were reprobed with anti-ALK (middle panels) or anti-Shc (lower panels) antibodies. The positions of ALK (220 and 140 kDa) and three isoforms of Shc (66, 52, and 44 kDa) are indicated by the arrowheads and bracket, respectively. The dot shows the position of Shc (66 kDa). (B,C,D,E) Lysates prepared as above were immunoprecipitated with antibodies as indicated and immunoblotted with anti-PY antibody (upper panels) and then reprobed with the indicated antibodies (lower panels). The positions of the immunoprecipitated proteins are indicated by dots. Asterisks in C show unidentified co-immunoprecipitated proteins with anti-p85 antibody.
- Fig. 5.
Effects of mAb16-39 on DNA synthesis. Serum-deprived 2-C4, parental NIH 3T3 (A), or SK-N-SH (B) cells were cultured for 2 days in the presence of various concentrations (nM) of normal rat IgG, mAb16-39, NGF, or 5% serum as indicated. (–) in the serum column indicates 0.1% serum. [3H]thymidine uptake over the last 4 hours of culture was measured and is presented as the mean±s.e.m. of three experiments performed in duplicate.
- Fig. 6.
Mitogenic properties of mAb16-39 are dependent on ERK activity. (A) Effect of mAb16-39 on ERK activation. 2-C4 cells were serum starved for 24 hours and treated with 4 nM normal rat IgG (–) or mAb16-39 (+) for 5 minutes or 3 hours. Equal amounts of total cell lysate were probed with phospho-specific anti-ERK1/2 antibody (ERK1/2-P). The blots were stripped and reprobed with anti-ERK antibody (ERK2). (B) Quantification of data presented in A. The intensity of the bands in the anti-phospho-ERK1/2 blot was normalized against the amounts of MAPK and expressed as fold activation over basal levels (IgG-treated). (C) Effect of the MEK inhibitor PD98059 on the mitogenic activity induced by mAb16-39. Serum-deprived 2-C4 cells were left untreated or were treated with 4 nM IgG or mAb16-39 for 2 days in the presence or absence of PD98059 (50 μM) or wortmannin (100 nM). [3H]thymidine uptake assay was performed as in Fig. 5, and the data are presented as the mean±s.e.m.
- Fig. 7.
mAb16-39 induces neurite outgrowth in an ERK-dependent manner. SK-N-SH cells were cultured in insulin, transferrin and selenium-supplemented media in the presence of mAb16-39 (4 nM), control normal rat IgG (4 nM), or NGF (2 nM) and photographed at day 2.5. Treatment of cells with mAb16-39 (C) resulted in extensive outgrowth of long neurites and increased cell number. Similar but less significant effects were observed in cells treated with NGF (B). Cells treated with control IgG (A) showed no significant effect on morphology. Neurite extension induced by mAb16-39 was efficiently blocked by the MEK inhibitor PD98059 (50 μM) (D). Scale bar, 50 μm. (E) Cell differentiation was scored as described in Materials and Methods and expressed as the mean±s.e.m. (%).
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