Journal: eLife

Article Title: FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase

doi: 10.7554/eLife.09811

Figure Lengend Snippet: ( A ) Schematic overview of anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) protein domain structure. ( B ) Alignment of the intracellular region of ALK and LTK, highlighting in yellow the Y1278 phosphoepitope in the activation loop which shares significant homology between ALK and LTK, and the Y1604 phosphoepitope of ALK which is not found in LTK. DOI: http://dx.doi.org/10.7554/eLife.09811.005

Article Snippet: The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK (for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technology), anti-ALK mAb135 (1:2000; [ ]), anti-pERK5 (1:1000; Cell Signaling Technology), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A (1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting; 16B12, Covance).

Techniques: Activation Assay

Journal: eLife

Article Title: FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase

doi: 10.7554/eLife.09811

Figure Lengend Snippet: ( A ) Neurite outgrowth in PC12 cells expressing either vector control or anaplastic lymphoma kinase (ALK) were cultured in medium from Human Embryonic Kidney (HEK) 293 cells transfected with either FAM150A or FAM150B, quantified below. Experiments were performed in triplicate and each sample within an experiment was performed in duplicate. Values represent mean ± SD from at least three independent experiments. ( B ) Whole cell lysates from PC12 cells expressing either vector control or ALK stimulated with medium from HEK293 cells transfected with vector control, FAM150A or FAM150B were analyzed by immunoblot. Analysis was carried out in the presence or absence of 250 nM crizotinib. Detection of ALK activation was visualized with pALK-Y1604 (arrowheads), ALK and pERK1/2 in whole cell lysates. The presence of FAM150A in supernatants was confirmed with anti-FAM150A antibodies, while the presence of FAM150B-HA was confirmed with anti-HA antibodies. Pan-ERK was employed for equal loading. ( C ) IMR32 cells harboring a wild-type ALK receptor were stimulated for 20 min with medium from HEK293 cells transfected with either vector control, FAM150A or FAM150B prior to analysis by immunoblot. Analysis was carried out in the presence or absence of 250 nM crizotinib. Stimulation with the ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowheads) and pERK1/2. Pan-ERK was employed for equal loading. ( D ) IMR32 cells harboring a wild-type ALK receptor stimulated with increased amounts of recombinant His-tagged FAM150A purified from Sf21 cells. Detection of ALK activation was visualized with ALK, pALK-Y1278 (arrowheads) and pERK1/2. Pan-ERK was employed for equal loading. ( E ) Time course of IMR32 cells stimulated with FAM150A conditioned medium. Stimulation with ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowheads), pERK5 and pERK1/2. Pan-ERK was employed for equal loading. ( F ) Time course of IMR32 cells harboring a wild type ALK receptor stimulated with FAM150B conditioned medium. Stimulation with ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowheads), pERK5 and pERK1/2. Pan-ERK was employed for equal loading. DOI: http://dx.doi.org/10.7554/eLife.09811.006

Article Snippet: The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK (for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technology), anti-ALK mAb135 (1:2000; [ ]), anti-pERK5 (1:1000; Cell Signaling Technology), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A (1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting; 16B12, Covance).

Techniques: Expressing, Plasmid Preparation, Cell Culture, Transfection, Western Blot, Activation Assay, Positive Control, Recombinant, Purification