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. 2022 Feb 14;23(4):e52904. doi: 10.15252/embr.202152904

Figure 2. 4D7 disrupts interaction with TpoR and downstream signalling.

Figure 2

  1. Cell extracts blotted for phospho‐STAT5, total STAT5, phospho‐ERK, total ERK and actin from TF‐1 TpoR cells after incubation with 10 or 20 µg/ml 4D7 or IgG for 4 or 8 h, in presence of TPO. The last line indicates TPO withdrawal.
  2. Similar experiment using TPO‐independent TF‐1 TpoR CALRdel61 cells.
  3. Similar experiment using TPO‐independent TF‐1 TpoR CALRdel52 cells at 8 h.
  4. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 h. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.
  5. Fraction of apoptotic sub‐G0 population of TF‐1 TpoR CALRdel61 cells after 48 h of 4D7 or IgG treatment (n = 3 biological replicates, bars represent standard deviation for three replicates, normalized to IgG, with a Student's unpaired t‐test used to determine statistical significance).
  6. Western blot showing caspase 3 cleavage occurring within 24 h of TPO withdrawal in TPO‐dependent TF‐1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 h of treatment with 20 µg/ml 4D7 in TF‐1 TpoR CALRdel61 and TF‐1 TpoR CALRdel52 cells.
  7. Western blot of TpoR immunoprecipitation under non‐reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF‐1 TpoR CALRdel61 disrupted by 8‐h treatment with 20 µg/ml 4D7 but not PBS or 20 µg/ml IgG. CALR monomers and dimers are detectable by polyclonal anti‐wild‐type CALR or anti‐mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wild‐type CALR protein; asterisk, non‐specific bands.

Source data are available online for this figure.