Figure EV3. In‐depth analysis of IQDMA shows its multi‐kinase inhibitory function.

• A
  Thermal shift profiles of 2 μM STAT5B with 100 μM IQDMA or 100 μM STAT5‐binding peptide (positive control). The local maxima indicate the melt temperature of the protein (T_m ± 0.3°C). One representative out of two independent experiments performed in technical triplicates is shown.
• B
  Fluorescence polarization assay using 180 nM STAT5B with 100 μM IQDMA or 100 μM peptide (positive control). The positive control exhibits a single‐site displacement profile. Error bars represent mean ± SD. Three independent experiments were performed.
• C
  IQDMA structure.
• D
  Subcellular fractions of SeAx cells treated with IQDMA for 18 h and immunoblotted for pY‐STAT3 (phospho‐Tyr (705)‐STAT3) and total STAT3. The cells were collected 30 min after 5 ng/ml IL‐2 cytokine addition. α‐Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho‐ and total STAT3 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One experiment was performed.
• E
  Bar graph showing the cellular pathways that are most affected by IQDMA treatment as determined by network enrichment analysis.
• F
  Subcellular fractions of SeAx cells treated with FRAx597 for 18 h and immunoblotted for pY‐STAT3/pY‐STAT5 levels (i.e., phospho‐Tyr (705)‐STAT3 and phospho‐Tyr (694/699)‐STAT5A/B) and total STAT3/5 levels. The cells were collected 30 min after 5 ng/ml IL‐2 cytokine addition. α‐Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho‐ and total STAT3/5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One experiment was performed.

Source data are available online for this figure.