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. 2014 May 23;289(28):19747–19757. doi: 10.1074/jbc.M114.547273

FIGURE 6.

FIGURE 6.

PDGFRβ, TβRI, and CD44 all bind each other, but the complex between PDGFRβ and TβRI is not dependent on CD44. A, Cos1 cells were transiently transfected with PDGFRβ-HA, TβRI-FLAG, and CD44–6myc or correspondingly tagged empty vectors. After 48 h, cell lysates were immunoprecipitated (IP) using FLAG, HA, or Myc antibodies. Proteins were released from beads by boiling in reducing sample buffer and subjected to SDS-PAGE and immunoblotting (IB) with antibodies against CD44, HA, and FLAG or GAPDH as loading control. Total cell lysates (TCL) were analyzed in parallel. B, Cos1 cells were transiently transfected with CD44 siRNA for 24 h and then with TβRI-FLAG and PDGFRβ-HA vectors for another 48 h. Cells were lysed and immunoprecipitated with FLAG antibodies, separated by SDS-PAGE, and detected by immunoblotting for CD44 and the HA and FLAG tags. C, BJ-hTERT foreskin fibroblasts were grown in 8-well chambers and transiently transfected with siRNA against CD44 or a scrambled control. Following starvation, cells were stimulated for 7 min with PDGF-BB (20 ng/ml), 2 h with hyaluronan (200 μg/ml), 1 h with TGFβ (1 ng/ml), or 1 h with 10% FBS. Cells were fixed and PLA was performed with mouse anti-PDGFRβ and rabbit anti-TβRI antibodies, followed by anti-mouse and anti-rabbit PLA probes conjugated with priming and nonpriming oligonucleotides. F-actin was stained with FITC-conjugated phalloidin. Single protein-protein interactions were visualized by fluorescence microscope as red dots. PLA signals per cell were quantified with Duolink Image Tool according to the manufacturer's instructions. Average is indicated in the graph by horizontal lines. D, Cos1 cells were transfected with PDGFRβ-HA and TβRI-FLAG, in combination with varying amounts of CD44-myc (0.1, 0.5, or 2 μg/sample) or empty vector. Cells were lysed, and immunoprecipitated with FLAG antibody and subjected to SDS-PAGE. The samples were then immunoblotted with specific antibodies against the FLAG and HA tags and CD44. Total cell lysates were run in parallel. Representative experiments out of three independent experiments are shown.