Figure 5.
DMF impairs the activity of JAKs. (A) The ABC DLBCL cell line OCI-Ly10 was treated with 40 μM DMF for the specified times and the phosphorylation of STAT3, STAT1, and IκBα was assessed by immunoblot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (B-C) ABC DLBCL cell lines were treated with solvent, 40 μM DMF, and/or 3 ng/mL recombinant human IL-6 (B) or 1 ng/mL interferon-α (IFN-α) (C), as indicated. Phosphorylation of STAT1/3 was visualized by immunoblot analysis. GAPDH served as loading control. (D) Analysis of JAK1 and TYK2 autophosphorylation in solvent-, IFN-α–, and/or DMF-treated ABC DLBCL cell lines. Arrowhead indicates phosphorylated TYK2 (P-TYK2). GAPDH served as loading control. (E) HBL-1 cells treated with solvent, 40 μM DMF, or 40 μM DMS were lysed, and reactive cysteine-containing proteins were labeled with biotin-coupled iodoacetamide (IA-biotin), isolated using streptavidin (SA) agarose, and analyzed by immunoblotting. (F) Schematic representation of JAK1 domain structure including FERM domain, Src homology 2 (SH2) domain, and kinase domains. Alignment of JAK1 sequences surrounding the conserved C257 from various species. (G) Using PDB entry 5IXI, human JAK1 was pictured around the modified cysteine residue (C257) to emphasize its solvent accessibility. The sulfur atom of the cysteine side chain is marked by a pink sphere. The protein chain (gray) is shown in cartoon representation. The close proximity of C257 to the receptor (dark blue) binding site is shown. (H) HEK293T cells were transiently transfected with V5-tagged IL-10 receptor (α subunit; IL10RA-V5) alone or in combination with Strep-tagged JAK1. Twenty-four hours after transfection, cells were treated with solvent or DMF for 1 hour and lysed; JAK1 was pulled down using Strep-Tactin beads. The interaction between IL10RA-V5 and JAK1-Strep was visualized by immunoblot analysis. (I) HBL-1 cells expressing control vector, FLAG-RelA, or FLAG-STAT3C were treated daily with solvent or 20 μM DMF in combination with recombinant human IL-6 and IL-10 (3 ng/mL each), as indicated. Cell numbers were determined after 48 hours and normalized to the respective controls lacking DMF. Data are representative of ≥2 (E,I) or 3 (A-D,H) independent experiments. Statistical significance was calculated using the Student t test. **P < .01.

DMF impairs the activity of JAKs. (A) The ABC DLBCL cell line OCI-Ly10 was treated with 40 μM DMF for the specified times and the phosphorylation of STAT3, STAT1, and IκBα was assessed by immunoblot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (B-C) ABC DLBCL cell lines were treated with solvent, 40 μM DMF, and/or 3 ng/mL recombinant human IL-6 (B) or 1 ng/mL interferon-α (IFN-α) (C), as indicated. Phosphorylation of STAT1/3 was visualized by immunoblot analysis. GAPDH served as loading control. (D) Analysis of JAK1 and TYK2 autophosphorylation in solvent-, IFN-α–, and/or DMF-treated ABC DLBCL cell lines. Arrowhead indicates phosphorylated TYK2 (P-TYK2). GAPDH served as loading control. (E) HBL-1 cells treated with solvent, 40 μM DMF, or 40 μM DMS were lysed, and reactive cysteine-containing proteins were labeled with biotin-coupled iodoacetamide (IA-biotin), isolated using streptavidin (SA) agarose, and analyzed by immunoblotting. (F) Schematic representation of JAK1 domain structure including FERM domain, Src homology 2 (SH2) domain, and kinase domains. Alignment of JAK1 sequences surrounding the conserved C257 from various species. (G) Using PDB entry 5IXI, human JAK1 was pictured around the modified cysteine residue (C257) to emphasize its solvent accessibility. The sulfur atom of the cysteine side chain is marked by a pink sphere. The protein chain (gray) is shown in cartoon representation. The close proximity of C257 to the receptor (dark blue) binding site is shown. (H) HEK293T cells were transiently transfected with V5-tagged IL-10 receptor (α subunit; IL10RA-V5) alone or in combination with Strep-tagged JAK1. Twenty-four hours after transfection, cells were treated with solvent or DMF for 1 hour and lysed; JAK1 was pulled down using Strep-Tactin beads. The interaction between IL10RA-V5 and JAK1-Strep was visualized by immunoblot analysis. (I) HBL-1 cells expressing control vector, FLAG-RelA, or FLAG-STAT3C were treated daily with solvent or 20 μM DMF in combination with recombinant human IL-6 and IL-10 (3 ng/mL each), as indicated. Cell numbers were determined after 48 hours and normalized to the respective controls lacking DMF. Data are representative of ≥2 (E,I) or 3 (A-D,H) independent experiments. Statistical significance was calculated using the Student t test. **P < .01.

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