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. 2015 Sep 29;4:e09811. doi: 10.7554/eLife.09811

Figure 2.  Conditioned medium containing either FAM150A or FAM150B activates endogenous ALK.

(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

Figure 2.

Figure 2—figure supplement 1. FAM150 proteins interact with heparin.

Figure 2—figure supplement 1.

(A) Purified FAM150A-HIS was incubated with heparin-agarose overnight at 4º°C prior to extensive washing. Bound proteins were eluted, separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with anti-FAM150A. Purified FAM150A-His (5 µg) was employed as input control. (B) Conditioned media from cells transfected with pcDNA3 control, pTT5-FAM150A-HA, or pcDNA3-FAM150B-HA was incubated with heparin-agarose for 3 hr at 4º°C prior to extensive washing. Bound proteins were eluted, separated on 13% SDS-PAGE and analyzed by immunoblotting with anti-HA. Conditioned media (50 µl) was employed as input control. *blue asterisk indicates FAM150A-HA, *red asterisk indicates FAM150B-HA in conditioned medium input.
DOI: http://dx.doi.org/10.7554/eLife.09811.007

Figure 2—figure supplement 2. Investigation of the effect of Heparin and FAM150A on ALK activation.

Figure 2—figure supplement 2.

(A) IMR32 cells were treated with FAM150A alone in the presence or absence of 10 µg/ml heparin alone for the times indicated, prior to analysis by immunoblot. Anaplastic lymphoma kinase (ALK) signaling was visualized with pERK1/2. Tubulin was employed for equal loading. (B) NB1 cells were treated with FAM150A alone or heparin alone or with FAM150A and heparin together at the indicated concentrations for 10 min, prior to analysis by immunoblot. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowhead) and pERK1/2. Pan-ERK was employed for equal loading.
DOI: http://dx.doi.org/10.7554/eLife.09811.008