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F. Kramer et al.
expression of both PDGFRb and PDGF-B in the bone mar- row of 15-month-old Gata-1low mice and suggests an increased activation of intracellular signaling in the overt fibrotic stage. To further analyze the activation status of the receptor, we applied a PLA combining PDGFRb and phosphotyrosine-targeting primary antibodies to analyze PDGFRb phosphorylation in situ (Figure 5C). Surprisingly, we did not observe increased PDGFRb tyrosine phospho- rylation at any stage of myelofibrosis in Gata-1low mice (Figure 5D, original PLA images are shown in Online Supplementary Figure 5). To verify these results, we per-
formed both an enzyme-linked immunosorbent assay (ELISA) with isolated protein from fresh frozen material and in situ staining for PDGFRb phosphorylation at Y751. In line with the PLA data, no differences in tyrosine phos- phorylation were detectable between the WT animals and Gata-1low mice at all ages (data not shown). These results suggest the presence of further counter-regulatory mecha- nisms, such as PTP. Therefore, to investigate the contribu- tion of the different PTP regarding the PDGFRb phospho- rylation status, we screened RNAseq data from 10-month-old Gata-1low mice and WT controls for differ-
AB
C
D
Figure 6. Expression and interaction dynamics of the phosphatase T-cell protein tyrosine phosphatase (TC-PTP) and platelet-derived growth factor receptor b (PDGFRb) in the bone marrow of Gata-1low mice at 5 months (5 M), 10 months (10 M) and 15 months (15 M) of age. (A) Quantitative analyses of TC-PTP protein expression by single recognition proximity ligation assay (PLA) in the bone marrow of Gata-1low mice and age-matched wild-type (WT) controls. (B) Representative images showing femoral bone marrow of Gata-1low mice stained for PDGF receptor b (PDGFRb, green) and TC-PTP (red). Nuclei were counterstained with DAPI (blue), scale bars=20 mm. (C) Illustration of a PLA for the analysis of PDGFRb–TC-PTP interaction. (D) Quantitative analysis of PDGFRb–TC-PTP interaction by PLA in the bone marrow of Gata-1low mice and age-matched WT controls, n=6 mice per group. 4051-9101 nucleated cells per mouse were analyzed for the presence of rolling circle products (RCP). *P≤0.05 versus the control group by Student t-test.
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haematologica | 2020; 105(8)