Fig. 2

SNCG protein is associated with β1 integrin and activates β1 integrin. a-b. Coimmunoprecipitation. Cell membrane proteins of HCT116 cells were collected and subjected to immunoprecipitated (IP) using anti-SNCG (a), anti-SNCG or anti-β1 integrin antibody (b). The IP proteins or total cell lysates were analyzed by Western blot. Normal IgG served as the negative control. c. Far-Western blot analysis. HCT116 cells were transfected with control siRNA (lane 1-2), and specific siRNA-β1-2 (lanes 3-4) for 48 h. Cells were treated without (lane 1, 3) or with 1 μmol/L rhSNCG (lane 2, 4). Cell lysates were subjected to SDS-PAGE and transferred to NC membrane. β1 integrin (prey protein) on the membrane is detected with SNCG (bait protein). More SNCG was associated with membrane β1 integrin in SNCG-treated cells than that in the control cells (lane 1, 2). Correspondingly, less SNCG were detected in β1 integrin knock-down cells than that in control cells (lane 2, 4). d-e, Effect of concentration and time treatment of SNCG on activated β1 integrin. HCT116 cells were stimulated with GST or GST-SNCG at various concentrations for 60 min (d) or at fixed concentration (1 μmol/L) for various times (e). Cell lysates were analyzed with the HUTS-21 mAb recognizing the activated form of β1 integrin. f, GST-SNCG treatment (1 μmol/L) upregulated activated β1 integrin subunit in HCT116 and SW480 cells. g, Colocalization of SNCG with F-actin. HCT116 cells grown on coverslips were transiently transfected with control siRNA or β1-specific siRNA-2. After 72 h, cells were treated with GST or GST-SNCG (1 μmol/L) for 60 min. Cells were fixed and stained with anti-SNCG (red) and FITC-Phalloidin (green). Colocalization of SNCG and F-actin was shown in yellow. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm