Figure 4. LIF‐activated STAT4 is phosphorylated on SPXX motifs within the C‐terminal transcription regulation domain.

• A
  Immunoblot analysis of STAT4 phosphorylation in CD4⁺ T cells pretreated with PBS or LY2784544 (1 μM) for 12 h followed by LIF for the indicated number of minutes.
• B
  Immunoblot analysis of CD4⁺ T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti‐LIFR antibody.
• C
  Coimmunoprecipitation and immunoblot analysis of LIF‐treated HEK293T cells cotransfected with the indicated constructs. The red asterisks denote phosphorylated STAT bands.
• D
  Sequence alignment showing that the C‐terminus of STAT4 and LIFR contains four SP repeats in both humans and mice. The SPXX motifs are displayed and underlined in red. The red asterisks point out the potential phosphorylated serine residues.
• E
  The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C‐terminus.
• F
  Immunoblot analysis of the indicated protein modifications in the total lysate of CD4⁺ T cells treated with LIF (20 ng/ml) at different time points.
• G–I
  Coimmunoprecipitation and immunoblot analysis of LIF‐treated HEK293T cells cotransfected with the indicated constructs.
• J
  Immunoblot analysis of the distribution of the indicated proteins in the CD4⁺ T‐cell fraction.
• K
  Cultured HeLa cells transfected with GFP‐tagged STAT constructs were exposed to LIF and imaged 30 min later. Scale bar, 50 μm.

Source data are available online for this figure.