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: Food Science & Nutrition

Article Title: Dihydroquercetin Attenuates Silica‐Induced Pulmonary Fibrosis by Modulating the Gut Microbiota and the Serum Metabolites in Mice

doi: 10.1002/fsn3.71389

Figure Lengend Snippet: DHQ treatment attenuated silica‐induced pulmonary fibrosis in C57/BL6 mice. (A, B) DHQ treatment increased the body weight and decreased pulmonary index in silicosis model mice. The changes of body weight (C) The levels of pro‐inflammatory cytokines (IL‐1β, TNF‐α, and TGF‐β) in serum from different groups at day 21 were detected by ELISA assay. (D) Representative pictures (×200) of HE‐stained and Masson‐stained lung sections from mice on day 21 were shown. Bar = 100 μm. (E, F) The inflammation and fibrosis score numbers of 0–3, corresponding to the grades of –, +, ++, and +++, were evaluated by experienced pathologists in a blinded fashion. (G) Representative results of western blot for α‐SMA, collagen I and fibronectin in lung tissues and the quantification of results. Data are shown as mean ± SD. All experiments were repeated three times. # p < 0.05, ## p < 0.01 vs. the control group; * p < 0.05, ** p < 0.01 vs. the SiO 2 group.

Article Snippet: Sigma‐Aldrich provided the SiO 2 (Cat#S5631) particles (around 80% diameter 1‐5 μm), which were filtered through sedimentation following Stokes' law, underwent acidic hydrolysis, and were baked overnight at 200°C for 16 h. Interleukin‐1β (IL‐1β) (CSB‐E08054m), tumor necrosis factor‐α (TNF‐α) (CSB‐E04741m), and transforming growth factor‐β (TGF‐β) (CSB‐E04726m) were acquired from Cusabio Biotechnology in Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Control

Journal: International heart journal

Article Title: Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1.

doi: 10.1536/ihj.16-572

Figure Lengend Snippet: Figure 2. Reduction of TGFβ1 expression by downregulation of RyR2in cardiomyocytes under mechani- cal stretch. A: Measurement of RyR2 mRNA level in cultured cardiomyocytes by Real-Time PCR. B: Expres- sion of RyR2 protein level in cardiomyocytes with Western Blotting. C: TGFβ1 in supernates of cultured cardiomyocytes was detected with ELISA analysis. D: Expression of TGFβ1 protein level in cardiomyocytes with Western Blotting. E: Measurement of tgfb1 mRNA level in cultured cardiomyocytes using Real-Time PCR. Values are expressed as mean ± SEM obtained from 3 independent experiments. shRyR2 indicates RyR2 knockdown lentiviral particle-infected cardiomyocytes; MS, cardiomyocytes under mechanical stretch; MS + ryr2 KD, RyR2 knockdown cardiomyocytes under mechanical stretch. *P < 0.05 versus control.

Article Snippet: ELISA assay: Supernates of cultured cardiomyocytes were collected and examined by Rat Transforming Growth Factor β1 ELISA Kit (CUSABIO BIOTECH, China) following the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Infection, Control