Fig 1: Internalization, trafficking and recycling of CXCR3-A. a Membrane or perinuclear localization of CXCR3 (λ = 488 nm) and colocalization with LRP1 (λ = 647 nm) in non-stimulated (NS) U87-CXCR3-A cells and upon stimulation with PS372424 at 15 and 60 min. Internalization and recycling of CXCR3 studied by immunostaining for clathrin, EEA1, GOPC, or Syntaxin6 (for each marker λ = 547 nm). Nuclei were stained with DAPI (λ = 405 nm). Scale: 5 µm. The experiments have been repeated three times with identical results. b Electron microscopy with immunogold labeling of CXCR3 (15 nm diameter) and Syntaxin6 (6 nm diameter) in ultrathin cryosections of U87-CXCR3-A cells stimulated with PS372424 for 60 min. TG trans-Golgi (n = 3 grids)
Fig 2: BLVRB loss reduces the membrane expression of HER2 and transferrin receptors.(A) Immunoblot (20 mg/lane) demonstrates loss of HER2 protein and its phosphorylation (Y1221/Y1222) in all BLVRB−/− clones; Actin is shown as a loading control. (B) ErbB2 Q-PCR of BLVRB+/+ or BLVRB−/− cells is presented as mean ± SEM normalized to actin (Actb, N = 3/cohort); p-value (not significant, N.S.) using unpaired t-test. (C) Confocal microscopy demonstrates loss of total and cell-surface HER2 in BLVRB−/− compared to BLVRB+/+ cells; size marker (20 mm) is shown. (D) Subcellular isolation and fractionation demonstrate enriched membrane HER2, with evidence for both cytoplasmic (predominant) and membrane-bound BLVRB in BLVRB+/+ cells; note the clear loss of total and membrane-bound HER2 in BLVRB−/− cells occurring in conjunction with EE1A membrane translocation (20 mg lysates/lane). Ponceau S protein staining as a loading control. (E) ER stress induced by 1 mM thapsigargin (TG) leads to exaggerated time-dependent HER2 loss in BLVRB−/− compared to BLVRB+/+ cells (20 mg lysates/lane); Hsc70 is used as the loading control. (F) Immunoblot (20 mg/lane) demonstrates downregulation of transferrin receptor (CD71) in BLVRB−/− compared to BLVRB+/+ cells, with concomitant increase in caveolin and EE1A, but not RAB7 and clathrin. (G, H) Flow cytometric analyses of BLVRB+/+ and BLVRB−/− cells (fixed, non-permeabilized) show cell-surface quantification of HER2 and CD71 abundance (mean fluorescence intensity (MFI), Panel G) or percent positivity (Panel H); data are presented as mean ± SEM from N = 5 – 6 technical and biologic replicates; p-values are shown; N.S. not significant. (I) Pan-phosphotyrosine antibody immunoblot reveals a global decrease in RTK activity across all BLVRB knockout clones. (J) Flow cytometric analyses of T47D/BLVRB+/+ and T47D/BLVRB−/− cells (fixed, non-permeabilized) show cell-surface quantification of CD71 (TfR) abundance (mean fluorescence intensity (MFI), left panel) or percent positivity (right panel); data are the mean ± SEM from N = 3 technical and biologic replicates; p-values are shown; N.S. not significant. (K) Box plots (displaying the median, upper quartile, minimum, and maximum values, Panel I) and corresponding ROC (receiver operator characteristic) curve (Panel K), demonstrate that elevated BLVRB expression in human breast cancer is associated with a worse pathological complete response (pCR) to HER2 targeted (Lapatinib and Trastuzumab) therapies; graphs and statistics were generated in silico [57].
from Cell Signaling Technology for Endosomal Marker Antibody Sampler Kit