Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: SMAD4 status and TGFβ 1 response of selected tumor cell lines were: (1) confirmed by PCR sequencing (data not shown) and (2) by [ 3 H] thymidine incorporation assays (data not shown). WT denotes wild type.

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Sequencing, Mutagenesis

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: Phosphorylation and immobilization of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored α V β 6 was immunoprecipitated after TGFβ 1 stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A) or Western blotting after biotinylation of all proteins and streptavidin detection (B). Presence of TGFβ 1 (C), α V and β 6 integrin (D) in the co-precipitates is also demonstrated. TGFβ-receptor-I and II (TGFβRI and TGFβRII) are expressed at nearly equal levels in all cell lines as demonstrated by western blotting from whole cell extracts (E). In part the cells were preincubated with α V - and β 6 -antibodies (1:100 each for 30 min) or with a TGFβ antibody (15 μg/ml for 30 min).

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Immunoprecipitation, Western Blot

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: Enhanced Tyrosine Phosphorylation of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored α V β 6 was immunoprecipitated after TGFβ 1 and/or fibronectin stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A). Reprobing with α V and β 6 antibodies show equal anounts of precipitates used (B).

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Immunoprecipitation, Western Blot

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: p125 FAK activation by mature TGFβ 1 via integrin α V β 6 . Stimulation of BxPC-3 with mature TGFβ 1 (10 nM for 10 minutes), immunoprecipitation with α V - and β 6 integrin antibodies after preparation of the cytoskeleton, followed by probing with pp125 Fak and p125 FAK antibodies. In part the cells were preincubated with α V - and β 6 -antibodies (1:100 each for 30 min), with a TGFβ antibody (15 μg/ml for 30 min), cytochalasin D and BAPTA AM, respectively.

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Activation Assay, Immunoprecipitation

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: Cell cycle genes in response to TGFβ 1 . Western Blot analysis of HeLa cells stimulated with 10 nM of mature TGFβ 1 for the time indicated. Cytoskeletally anchored proteins are differentially marked. In part the cells were preincubated with α V - and β 6 -antibodies (1:100 each for 30 min), with a TGFβ-RII antibody (15 μg/ml for 30 min), cytochalasin D, BAPTA AM and MEK1 inhibitor PD98059, respectively.

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Western Blot

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: Enhanced level of cytoskeletal anchored proteins in response to TGFβ 1 (A). Western Blot analysis of BxPC-3 and HeLa cells as indicated after stimulation with TGFβ 1 for the time indicated. Cytoskeletally anchored proteins are differentially marked. In part the cells were preincubated with α V - and β 6 -antibodies (1:100 each for 30 min), with a TGFβ-RII antibody (15 μg/ml for 30 min), cytochalasin D, BAPTA AM and MEK1 inhibitor PD98059, respectively. Purity of the TGFβ 1 used (B). Ten nanogram of mature TGFβ 1 and latent TGFβ 1 were subjected to non-reducing SDS-PAGE dollowed by silver staining. No latant TGFβ 1 could be detected in the mature TGFβ 1 used for stimulation. BxPC-3 cells are SMAD4 -/- (C). One hundred microgram of whole cell extract from BxPC-3 and HeLa cells were probed with p125 FAK and SMAD4 antibodies on the same membrane. As reported, BxPC-3 cells are found to be SMAD4 -/- .

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Western Blot, SDS Page, Silver Staining

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: Activation and nuclear translocation of SMAD2/3 in response to TGFβ 1 (A). Nuclear and cytoplasmatic fraction of cellular proteins (BxPC-3) after stimulation with 10 nM of TGFβ 1 for 10 minutes and Western blot analysis for SMAD2/3 and phosphorylated SMAD2/3. Purity of cytoplasmic and nuclear fraction (B). Cytoplasmic and nuclear extracts from K562 cells were probed with p125 FAK , PCNA and Iκ Bα antibodies at the same time. As predicted, p125 FAK cold exclusively be detected in the cytoplasmic extract, whereas PCNA is found in the nucleus. Iκ Bα served as loading control.

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Activation Assay, Translocation Assay, Western Blot

Journal: Molecular Cancer

Article Title: TGFβ 1 signaling via α V β 6 integrin

doi: 10.1186/1476-4598-2-28

Figure Lengend Snippet: Hypothesis about an alternate TGFβ 1 signaling pathway via α V β 6 integrin, independent of RGD. This pathway may be required for full TGFβ 1 induced transcriptional activation, which explains the TGFβ 1 sensitivity of those cells lacking DPC4/SMAD4 function that still react with growth inhibition.

Article Snippet: The following antibodies were used in a dilution of 1:1,000: TGFβ 1 (Santa Cruz [sc], sc-146), p-Tyr (sc-7020), β 6 -integrin (sc-6632), α V -integrin (sc-6617), p125 FAK (sc-557), TGFβ 1 -RI (sc-402), TGFβ 1 -RII (sc-400-G), ERK1/2-P (sc-7383), SMAD2/3 (sc-6033), SOS1/2 (sc-259), p130 cas (UBI-06-500), PCNA (sc-56), p21 WAF1 (sc-6246), p27 KIP (sc-1641), c-fos (sc-7202), c-jun (sc-44), raf1 (sc-133), p21 Ras (sc-35) and phospho-threonine antibody (New England Biolabs, # 9381).

Techniques: Activation Assay, Inhibition

Journal: PLoS ONE

Article Title: S100A9 Interaction with TLR4 Promotes Tumor Growth

doi: 10.1371/journal.pone.0034207

Figure Lengend Snippet: A. Quantitative real time RT-PCR analysis (see ) of TGFβ RNA expression from CD11b + cells (>90% pure by FACS analysis) from the spleen of C57BL/6, S100A9 −/− and TLR4 −/− animals in the absence of, or 14 days after inoculation, with 50,000 EL4 lymphoma cells subcutaneously. The mean expression from 4 separate experiments is shown where the expression in the C57BL/6 controls have been set to 1. B. ELISA measurements of TGFβ serum levels of C57BL/6 (filled circles), RAGE −/− (filled triangles), S100A9 −/− (filled squares) and TLR4 −/− (filled diamonds) in the absence of, or 14 days after inoculation (open symbols), with 50,000 EL4 lymphoma cells subcutaneously. Statistical analysis using two-tailed t test *** p = 0.0008; * p = 0.039 and 0.0028, respectively. There was no statistical significant difference in TGFβ serum levels between EL4 inoculated C57BL/6 or RAGE −/− mice.

Article Snippet: The TGFβ1 Quantikine ELISA kit (Abnova Taipei, Taiwan) was utilized to determine serum TGFβ levels, as indicated.

Techniques: Quantitative RT-PCR, RNA Expression, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: S100A9 Interaction with TLR4 Promotes Tumor Growth

doi: 10.1371/journal.pone.0034207

Figure Lengend Snippet: A. Binding of S100A9 to immobilized TLR4/MD2 complex is blocked by ABR-215050. Sensorgrams obtained after injection (2 min at 30 µL/min) of 50 nM S100A9 ± ABR-215050 over amine coupled TLR4/MD2 (density ∼2.3 kRU). Sensorgrams from top to bottom: S100A9 without competitor and with 3.91, 31.25 and 1,000 µM ABR-215050. Arrows indicate injection of sample (1); sample buffer - i.e. HBS-P containing 1 mM Ca 2+ and 10 uM Zn 2+ (2); and regeneration of surface with 3 M EDTA (3). B. Anti-tumor effect of ABR-215050 in EL4 tumors inoculated (s.c.) into wild type mice. The ABR-215050 was administrated in the drinking water at 30 mg/kg/day seven days/week from day 0 throughout the experiment. Each data point represents mean ± SEM (n = 10; p<0.01, Mann Whitney U). Control animals received only normal drinking water. The water intake of the animals was not affected by the presence of ABR-215050 in the drinking water. C. ELISA measurements of TGFβ serum levels day 20 in C57BL/6 animals inoculated with EL4 tumors and animals treated with 30 mg/kg/day of ABR-215050, as indicated (p = 0.0037, Student t test).

Article Snippet: The TGFβ1 Quantikine ELISA kit (Abnova Taipei, Taiwan) was utilized to determine serum TGFβ levels, as indicated.

Techniques: Binding Assay, Injection, MANN-WHITNEY, Control, Enzyme-linked Immunosorbent Assay