Fig. 3

JAK2 controls proteolysis of PDGFRβ. a SUM159PT cells were reversed transfected with 1 μg of DNA of empty vector, MYC, mJAK2 or STAT3 using Lipofectamine 3000 for 24 h and PDGFRβ levels were determined by western blot. b Top: SUM159PT cells were reversed transfected with 1 μg of empty vector or mJAK2 for 24 h followed by 100 μg/ml cycloheximide (CHX) and cells were harvested at indicated time points. PDGFRβ and β-actin levels were determined by western blot. Bottom: Quantification of immunoblot images was performed using ImageJ software (NIH, Bethesda, MD, USA) and is presented in graphical form. The levels were normalized against β-actin, n = 3 with SEM. c SUM159PT cells were reversed transfected with 1 μg of empty vector or mJAK2 for 24 h followed by combination treatment with proteasomal (MG132) and lysosomal inhibitors (pepstatin and leupeptin) for 4 h. PDGFRβ levels were determined by western blot. d SUM159PT cells were reversed transfected with 1 μg of empty vector, mJAK2 wildtype or JAK2 kinase dead constructs for 24 h. PDGFRβ levels were determined by western blot. e SUM159PT cells were reversed transfected with 1 μg of empty vector, mJAK2 wildtype or JAK2 kinase dead constructs for 24 h followed by PDGF-BB (20 ng/ml) stimulation for 5 min. Phosphorylated PDGFRβ levels were determined by western blot. f HEK293T cells were reversed transfected with 1 μg of empty vector, mJAK2 wildtype or JAK2 kinase dead constructs for 24 h followed by combination treatment with proteasomal (MG132) and lysosomal inhibitors (pepstatin and leupeptin) for 4 h. Cell lysates were immunoprecipitated using either HA- or JAK2- specific antibodies and immunoblotted for indicated proteins. g Immunofluorescence analysis of PDGFRβ localization with JAK2. HEK293T cells were reversed transfected with wildtype or kinase dead mJAK2 constructs with GFP-tagged PDGFRβ expression constructs for 24 h, followed by combination treatment with proteasomal (MG132) and lysosomal inhibitors (pepstatin and leupeptin) for 4 h. Cells were fixed, permeabilized and stained with JAK2-specific antibody. h Sequence alignment of putative JAK2 consensus phosphorylation motif which recognizes YXX [L/I/V] in PDGFRβ. Possible Tyrosine sites are indicated in red font. i HEK293T cells were reverse transfected with wildtype and mutant PDGFRβ at indicated sites as shown in panel H in the absence and presence of mJAK2 wildtype or kinase dead construct. PDGFRβ levels were determined by western blot. j Heatmap analysis of correlation of PDGFRβ with JAK2 levels in TCGA breast cancer samples. Patient samples were divided into PAM50 subtypes. Data derived from cbioportal (http://www.cbioportal.org/). k Breast cancer TCGA patient’s data (http://tumorsurvival.org) was divided into two subgroups based on PDGFRβ and JAK2 expression and survival probability was plotted