Pdgfrβ

Primary cells were harvested in radioimmunosorbent assay (RIPA) buffer, tissue samples were snap-frozen in liquid nitrogen. Cells or tissues were disrupted using a TissueLyser (Qiagen) in RIPA buffer. Equal amounts of total cell protein were separated using NuPAGE® Novex 4%-12% Bis-Tris Mini Gels with MES SDS running buffer (Invitrogen) at 4 °C, transferred to Protran Nitrocellulose Blotting Membrane (GE Health Care Life Sciences) overnight at 4 °C, blocked with milk powder at room temperature (RT), washed 3× with Tris-buffered saline with 0.1% w/v Tween (TBST) at RT, incubated with primary antibodies (Lrp1: polyclonal LRP1-377, provided by J.Her.; PDGFRβ: Millipore 06-498; β-actin: Sigma, clone AC-15, 06-A5441) in TBST with 10% w/v bovine serum albumin (Sigma) overnight at 4 °C, washed 3× with TBST (RT), incubated with secondary horseradish peroxidase-labeled antibodies (Jackson ImmunoResearch) in TBST for 90 min at RT, washed 3× with TBST, and finally 1× with PBS. Immunoblots were developed with enhanced chemiluminescence (GE Health Care Life Sciences).

Standard Western blot analysis was performed on 10 μg of protein from the cell lines of interest using protocols as previously described [13 (link)]. PDGFRβ was purchased from Millipore (catalog # 06-495), PDGFRα (product # 9360) and GP130 from Cell Signaling Technology (product #3732), and α-IL6R from Thermo scientific (catalog #PA5-27975).