Journal: Oncogene

Article Title: IL-6 mediated activation of STAT3 bypasses Janus kinases in terminally differentiated B lineage cells.

doi: 10.1038/sj.onc.1205815

Figure Lengend Snippet: Figure 1 RT – PCR and Western analysis reveal lack of Jak1 ex- pression in most pristane induced plasmacytoma cell lines (a) RT – PCR analysis for RNA expression of Jak kinases in murine plasmacytoma cell lines. Total RNA was isolated from the indi- cated cell lines, reverse transcribed and cDNA’s were added to PCR reactions containing primers specific for Jak1, Jak2, Tyk2 and b-actin. PCR samples were subjected to 36 (Jak kinases) or 28 (b-actin) cycles of amplification. (b) Analysis of Jak1 protein expression in murine plasmacytomas. Immunoprecipitation was performed using 1 mg of total extracted protein from the indi- cated lines with antibody to Jak1 followed by blotting with the same antibody (upper panel). The same lysates were subjected to direct Western analysis with anti-Tyk2 and anti-STAT3 (lower panels). (c) Tyrosine phosphorylation of STAT3 in response to IL-6 in plasmacytoma cell lines lacking Jak1 protein. Serum- starved S107, X24, 1165 and 8.36 cells were treated with IL-6 (10 ng/ml) for 15 min. Cell lysates were immunoprecipitated with anti-STAT3 and blotted with anti-phosphotyrosine (4G10) anti- bodies. The membrane was then stripped and re-analysed with anti-STAT3

Article Snippet: The following antibodies were used for Western blot analysis: anti-Jak1 (Transduction Laboratories); anti-phosphotyrosine (PY99), anti-Jak2 (HR-758), anti-Tyk2 (C-20), anti-STAT3 (H-190) (Santa Cruz Biotechnology); anti-pTyr(705)STAT3, anti-pSer(727)STAT3, anti-pTyr(1054/ 1055)Tyk2, anti-pTyr(701)STAT1 (Cell Signaling); and antiphosphotyrosine 4G10 (Upstate Biotechology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, RNA Expression, Isolation, Reverse Transcription, Expressing, Immunoprecipitation, Phospho-proteomics, Membrane

Journal: Oncogene

Article Title: IL-6 mediated activation of STAT3 bypasses Janus kinases in terminally differentiated B lineage cells.

doi: 10.1038/sj.onc.1205815

Figure Lengend Snippet: Figure 3 Activation of Jak2 is not required for tyrosine phos- phorylation of STAT3 following IL-6 stimulation. (a) S107, X24, 8.36 and 1165 cells were either serum-starved (ST), treated with IL-6 (10 ng/ml) for 15 min (IL-6) or maintained un-starved in the continued presence of IL-6 (UST). Cell lysates (1 mg of to- tal protein for each cell line) were immunoprecipitated with anti- Jak2 and Western analysis was carried out with anti-phosphotyr- osine (4G10) antibodies. The membrane was then stripped and re- analysed with anti-Jak2. (b) Serum-starved 8.36 cells were pre- treated with solvent (D, dimethylsulphoxide) or AG490 at the concentrations indicated for 4 h and then stimulated with IL-6 (10 ng/ml) for 15 min. Serum-starved untreated cells were used as controls. Cell lysates were immunoprecipitated with anti- STAT3 and blotted with anti-phosphotyrosine (PY99) antibodies. The membranes were then stripped and re-analysed with anti- STAT3. (c) Serum-starved 8.36 cells were pretreated with solvent (D, dimethylsulphoxide) or AG490 (100 mM) for 4 h and then stimulated with IL-6 (10 ng/ml) for 15 min. Lysates from 8.36 cells were prepared and subjected to Western analysis using antibodies to pTyr(1054/1055)Tyk2. The membranes were then stripped and re-analysed with anti-Tyk2

Article Snippet: The following antibodies were used for Western blot analysis: anti-Jak1 (Transduction Laboratories); anti-phosphotyrosine (PY99), anti-Jak2 (HR-758), anti-Tyk2 (C-20), anti-STAT3 (H-190) (Santa Cruz Biotechnology); anti-pTyr(705)STAT3, anti-pSer(727)STAT3, anti-pTyr(1054/ 1055)Tyk2, anti-pTyr(701)STAT1 (Cell Signaling); and antiphosphotyrosine 4G10 (Upstate Biotechology).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Membrane, Solvent

Journal: Oncogene

Article Title: IL-6 mediated activation of STAT3 bypasses Janus kinases in terminally differentiated B lineage cells.

doi: 10.1038/sj.onc.1205815

Figure Lengend Snippet: Figure 4 Phosphorylation of Tyk2 is not sufficient for activation of STAT3. (a) Serum-starved S107, X24, 1165 and 8.36 cells were treated with IL-6 (10 ng/ml) for 15 min. Cell lysates were immu- noprecipitated with anti-Tyk2 and blotted with anti-phosphotyro- sine (4G10) antibodies. The membrane was then stripped and re- analysed with anti-Tyk2. (b) Dominant negative kinase-deficient mutant of Tyk2 does not affect IL-6-induced tyrosine phosphor- ylation of STAT3. Empty vector (V) transfected X24 cells and stably transfected X24-Tyk2 derivative cells (X24-Tyk2wt and X24-Tyk2D/N) were treated with IL-6 (10 ng/ml) for 15 min or untreated as indicated. Cell lysates were subjected to Western analysis using antibodies to Tyk2, pSer(727)STAT3 and pTyr(705)STAT3. The membrane was then stripped and re-ana- lysed with anti-STAT3

Article Snippet: The following antibodies were used for Western blot analysis: anti-Jak1 (Transduction Laboratories); anti-phosphotyrosine (PY99), anti-Jak2 (HR-758), anti-Tyk2 (C-20), anti-STAT3 (H-190) (Santa Cruz Biotechnology); anti-pTyr(705)STAT3, anti-pSer(727)STAT3, anti-pTyr(1054/ 1055)Tyk2, anti-pTyr(701)STAT1 (Cell Signaling); and antiphosphotyrosine 4G10 (Upstate Biotechology).

Techniques: Phospho-proteomics, Activation Assay, Membrane, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation, Transfection, Stable Transfection, Western Blot