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. 2015 Aug 10;112(34):E4717–E4725. doi: 10.1073/pnas.1514230112

Fig. 2.

Fig. 2.

Traptamers specifically bind and activate the PDGFβR. (A) Extracts prepared from C127 cells expressing MSCV vector (V), the E5 protein, or the indicated traptamer were immunoprecipitated with anti-PDGF receptor (PR) antibody and blotted for phosphotyrosine (PY, Upper) or PDGFβR (Lower). m, mature PDGFβR; p, precursor form of the receptor with immature carbohydrates activated by the E5 protein and some of the traptamers. (B) Extracts were prepared from human diploid fibroblasts that were untreated (MSCV), acutely treated with PDGF-DD (PDGF) or with a mixture of growth factors, or stably transformed by traptamer LI-5, LI-8, and LI-11. phospho-RTK arrays were incubated with cell extracts, processed, and exposed to film for the same length of time. The spots representing PDGFβR are underlined in red on all arrays. EGF receptor, PDGFαR, and Axl are underlined in blue, green, and purple, respectively, on the array probed with the extract of control cells. The dark pairs of spots at the corners are standards. (C) BaF3 cells expressing the PDGFβR (PR), the erythropoietin receptor (EpoR) (Left), or the c-kit (Right) were infected with MSCV or MSCV expressing the indicated traptamer or v-sis (for PR cells) or treated with EPO (for EpoR cells) or stem cell factor (SCF, for c-kit cells). The number of live cells was counted 5 d after IL-3 removal. Each experiment was performed three times. Although the inherent variability of this assay precluded a rigorous statistical analysis, the same trends were observed in each experiment. The graphs show the results of a representative experiment. (D) BaF3 cells expressing the LXSN empty vector, the PDGFβR, a chimeric PDGFβR containing the transmembrane domain of the PDGFαR (βαβ), or a mutant PDGFβR were infected with MSCV or MSCV expressing v-sis, the E5 protein, or the indicated traptamer. The number of live cells was counted 5 d after IL-3 removal. The experiment was performed three times and the same trends were observed in each experiment. The graph shows the results of a representative experiment. (E) Detergent extracts were prepared from untransformed C127 cells harboring MSCV (V) or cells transformed by the indicated FLAG-tagged (+) or untagged (−) traptamer. Extracts were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibody. (F, Left) Detergent extracts were prepared from BaF3 cells expressing the PDGFβR and MSCV or the indicated FLAG-tagged traptamer. US2 and US16 are inactive unselected clones. Extracts were imunoprecipitated with anti-FLAG and immunoblotted with anti-PDGF receptor. Both panels were from the same exposure from the same gel; an irrelevant lane was removed. (Right) Detergent extracts were prepared from BaF3 cells expressing the PDGFβR or the βαβ chimeric receptor together with MSCV or FLAG-tagged LI-5 or LI-7. Extracts were immunoprecipitated with anti-FLAG and immunoblotted with anti-PDGF receptor.