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. Author manuscript; available in PMC: 2010 Oct 20.
Published in final edited form as: Nat Genet. 2010 May 16;42(6):530–535. doi: 10.1038/ng.587

Figure 4. Identification of NUP214-ABL1 oncogene as a novel substrate of PTPN2.

Figure 4

  1. Desphosphorylation of NUP214-ABL1 was observed upon induced overexpression of wild type PTPN2 (PTPN2-WT). Immune precipitation (IP) of PTPN2-D182A resulted in co-precipitation of NUP214-ABL1 protein, showing a direct interaction between kinase and phosphatase.
  2. Viable cell numbers were determined 3 days after electroporation with respective siRNA’s. Decrease in PTPN2 protein levels resulted in a significant proliferative advantage of ALL-SIL cells.
  3. Cells were exposed to imatinib at time point of maximal PTPN2 knockdown. Western blot analysis showed differential activation status of STAT family members downstream of NUP214-ABL1. ERK1/2 and a SRC-family kinase were detected to be constitutively active in ALL-SIL cells and knockdown of PTPN2 caused an augmented phosphorylation which stayed unchanged upon imatinib treatment. Loading control: ERK1/2
  4. IP experiments of ALL-SIL cells after electroporation with siRNA’s revealed an enhancing effect on JAK3 kinase activation (upper) and identified the differentially activated SRC-family kinase as LCK. Phosphorylation of JAK3 and LCK were detected with a phosphotyrosine (pTyr) or phospho-SRC family antibody, respectively. JAK3 or LCK were used to ensure equal protein loading.
  5. Ba/F3 cells carrying indicated constructs were grown in absence of IL-3 and mean growth ± s.e.m. was recorded in triplicate for 8 days. No s.e.m. is shown in case of values below 0.03.
  6. Western blot analysis of NUP214-ABL1 dependent Ba/F3 cells demonstrated stronger constitutive activation of NUP214-ABL1 and STAT5 in case of concomitant shRNA-mediated knockdown of Ptpn2. Loading control: c-ABL, STAT5
  7. Induced overexpression of PTPN2-WT in Ba/F3 cells carrying the NUP214-ABL fusion results in dephosphorylation of the oncogenic kinase and its downstream mediator STAT5. C-ABL is shown as loading control.

Values shown below immunoblots represent normalized (for loading control) quantitative values. Open bars: siRNA PTPN2; closed bars: siRNA control; bars show average ± s.e.m. Error bars represent s.e.m. *p<0.05, **p<0.005.

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