Ab134123

SDS-PAGE of 25 μg of protein was performed on 8% reducing gels. Proteins were transferred at 4 °C onto PVDF membranes in tris-glycine buffer containing 20% methanol. Total protein loading was measured prior to blotting using Ponceau S staining and densitometric analysis. Non-specific binding sites were blocked with 5% skim milk in tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) at room temperature. The blots were thoroughly washed in TBS-T before applying primary antibodies overnight at 4 °C with shaking. Primary antibodies were used at the following dilutions: ED-A (cellular) fibronectin (1:1000; MAB1940; MilliporeSigma, Burlington, MA; or NBP1-91258; Novus), SMemb (1:1000; ab684; Abcam), αSMA (1:5000; A2547; Sigma). Because the primary fibroblasts originated from a heterogenous population of cells, vimentin (1:2000; ab8069; Abcam) and platelet-derived growth factor receptor alpha (PDGFRα; 1:1000; ab134123; Abcam) were used as a phenotype controls on each blot. Appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied at a 1:5000 dilution for 1 hour at room temperature. Protein detection was done using ECL substrate, and protein bands were visualized on blue X-ray film. Protein expression was measured by relative densitometry using Quantity One® analysis software (version 4.6.9; Bio-Rad).

The fourth mouse mammary gland of eight-weeks-old C57BL/6J mice were collected with skin and fixed with 10% buffered formalin. After embedding in paraffin, the cross-sections of the tissue were prepared and used for H&E staining and immunohistochemistry. Immunohistochemistry was performed by the Pathology Solid Tumor Core at City of Hope using Ventana Discovery Ultra IHC Auto Stainer (Roche Diagnostics, Indianapolis, IN, United States). Heat-mediated antigen retrieval was performed using Cell Conditioning Buffer 1 (Roche Diagnostics; pH 8.5) for an hour. Antibodies used for the immunostaining included: ERα rabbit polyclonal antibody (06–935, Millipore Sigma; 1:400), anti-PDGFRα rabbit monoclonal antibody (ab134123, Abcam, Cambridge, United Kingdom; 1:50), and anti-DPP4 rabbit monoclonal antibody (ab187048, Abcam; 1:500). Images were captured on the Zeiss Observer II (Carl Zeiss, Oberkochen, Germany; for H&E staining), VENTANA iScan HT (Roche Diagnostics; for PDGFRα and DPP4 immunohistochemistry) or Nano Zoomer S360 (HAMAMATSU PHOTONICS, Shizuoka, Japan; for ERα immunohistochemistry).