Fig. 8: Localization and expression of TrkB, panTrk and pTrk in grade IV glioblastoma.

A Nestin/TrkB-positive cells in glioblastoma. Sections were labeled for anti-Nestin, to identify glioblastoma, and the TrkB receptor. Confocal laser scanning images. DAPI was used as the counterstain. Some Nestin+ cells show a high abundance of TrkB (cyan arrows). B Immunolabeling of representative glioblastoma cryosections. with anti-Nestin and anti-pTrk-kin. Single Nestin+ cells show a high abundance of pTrk-kin (upper panel, yellow arrows). Second and third panels: High-resolution confocal image stack showing pTrk in intracellular, immunoreactive clusters. Lower panel. Membrane bleb-like structures (yellow arrow) in single cells with strong intracellular pTrk-kin label. C RT-qPCR revealed abundant expression of TrkB kinase transcript in grade IV glioblastoma. Normalization of TrkB kinase expression levels in relation to RNA polymerase II. Single data points, mean values, and standard deviations are shown. The amplicon sizes were verified by agarose gel electrophoresis, as indicated. D Immature phospho-active Trk kinase in glioblastoma. Western blotting of whole-cell lysates generated from frozen post-mortem glioblastoma samples with the indicated antibodies. Lanes 1–6 show the lysates generated from different tissue pieces of the same glioblastoma sample. Note the high abundance of panTrk kinase and pTrk in one tissue sample (lane 2). Lane 7–9 shows representative TrkB-positive, panTrk kinase-negative, pTrk-kin-negative glioblastoma samples. This sample (pat. 1 (rec)) is a recurrent cancer sample from the same patient. γ-Adaptin was labeled as loading control. E TrkB and phospho-active Trk kinase in glioblastoma cells. Western blotting of whole-cell lysates from human brain or glioblastoma samples. Lanes 1–5 show control brain samples (frontal brain). PanTrk kinase immunoreactivity was observed at 130 kDa, indicating mature Trk (either TrkA, B or C). Lanes 6–9 show total lysates from different grade IV glioblastoma biopsies. Note the high abundance of panTrk kinase and pTrk-kin at approximately 90 kDa, indicating immature, phosphorylated Trk. For all the lanes, 40 µg of total protein was blotted.