FIGURE 5.

SPRR3 interacts with PDGFRβ and integrin β1 and facilitates their interaction. A, Sprr3 ^−/− MEFs (P1‐P5) overexpressing either GFP or SPRR3 were used for immunoprecipitation (IP) of PDGFRβ, integrin β1 and SPRR3. Western blot analysis of the input cell lysates and IP eluate are shown. IP of PDGRβ was able to pull down integrin β1 only in the presence of SPRR3, as well as SPRR3 itself. Conversely, IP of integrin β1 was only able to pull‐down PDGFRβ in the presence of SPRR3, as well as SPRR3. SPRR3 pull‐down identified both integrin β1 and PDGFRβ in cells positive for SPRR3 (not GFP knockout cells). Negative isotype controls did not display any of the proteins after pull‐down. B, In situ proximity ligation of Integrin β1 and PDGFRβ showed enhanced receptor pairs in wild‐type MEFs compared to Sprr3 ^−/− MEFs (indicated by red). Secondary probes showed minimal staining. Scale bars represent 20 µm. Average interactions per cell (indicated by DAPI stain) per field were quantified. Statistical analysis was performed by t test where ***P < .001, n = 4‐7. C, WT and Sprr3 ^−/− MEFs (P1‐P5) were serum starved, then, plated on 1 µg/mL fibronectin coated plates in serum‐free media for 24 hours. Cells were treated with/without 10 µg/mL Integrin β1 antibody for 1 hour at 37°C prior to stimulation with PDGF. BrdU labeling was done for 18 hours and BrdU proliferation assay was performed as per manufacturer's instructions (Millipore). Statistical analysis was performed using a 2‐way ANOVA, where P < .05, n = 3