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Fig. 1 | Cell Communication and Signaling

Fig. 1

From: Syndecan-4/PAR-3 signaling regulates focal adhesion dynamics in mesenchymal cells

Fig. 1

Thy-1 treatment induced association of PAR-3 with Syndecan-4 in DI TNC1 astrocytes. a Cell lysates obtained upon FBS or (b) Thy-1-Fc-Protein A (Thy-1) stimulation were precipitated using biotinylated peptides consisting in the 10 amino acids of Syndecan-4 cytoplasmic domain (SDC4) or control peptides, in which the last 4 amino acids (EFYA) were replaced by glycine. Samples were immunoblotted for PAR-3. Syntenin was used as a precipitation control since it interacts directly with the Syndecan-4 EFYA sequence. c Syndecan-4-HA transfected DI TNC1 cells were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) as a negative control. Cell lysates were immunoprecipitated with the anti-HA antibody and immunoblotted for PAR-3, FAK, Syndecan-4, or Syntenin. d Association between PAR-3 and Syndecan-4 was evaluated by Proximity Ligation Assay (PLA). DI TNC1 cells were transfected with Syndecan-4-HA or with the empty plasmid. Subsequently, cells were serum starved for 30 min and stimulated or not (NS) with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL), fixed and processed for PLA using antibodies against PAR-3 or HA. Pictures are representative of PLA puncta observed for each condition as indicative of the interaction between PAR-3 and Syndecan-4-HA. Lower panels show magnification of the indicated area. Magnification bar = 10 μM. e Graph shows the number of PLA puncta per cell. ***P < 0.001

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