Journal: Scientific Reports

Article Title: Identification of chlorophyll a-b binding protein AB96 as a novel TGFβ1 neutralizing agent

doi: 10.1038/s41598-021-87454-x

Figure Lengend Snippet: VA aqueous extract [VA(aq)] contains an active TGFβ1 neutralizing agent. The effects of VA extract on endogenous ( a ) and exogenous ( b ) TGFβ1 levels in the supernatant of monocyte derived dendritic cells. n = 1 for a; n = 3 for b. ( c ) Unbound (remaining in supernatant after incubation) and bound (liberated after washing, acidification and neutralization) active TGFβ1 concentrations (starting conc.: 2 ng/ml) after incubation with VA(aq) for 2 h at room temperature (RT). Representative of 3 independent assays. Technical triplicates. Mean +/− SEM shown. ( d ) The unbound and bound IL-10, IL12, latent TGFβ1 and active TGFβ1 concentrations (starting conc.: 4 ng/ml) after incubation with VA(aq) for 2 h at RT. Technical triplicates. Mean +/− SEM shown. ( e ) The unbound and bound active TGFβ1 concentrations (starting conc.: 2 ng/ml) after incubation with sesquiterpene lactones and VA(aq) for 2 h at RT. Technical triplicates. Mean +/− SEM shown. ( f ) The levels of active TGFβ1 bound to aqueous extracts (1 mg/ml) from different species of plants [normalized to VA(aq)] when incubated with 2 ng/ml active TGFβ1. Species in order of x axis: (1) Vernonia amygdalina Del. (Compositae). (2) Vernonia arkansana DC. (Compositae). (3) Helianthus annuus L. (Compositae). (4) Aster dumosus Hoffm. (Compositae). (5) Campanula lactiflora M.Bieb. (Campanulaceae). (6) Petroselinum crispum (Mill.) Fuss (Apiaceae). (7) Ocimum basilicum L. (Lamiaceae). (8) Solanum lycopersicum L. (Solanaceae). (9) Spinacia oleracea L. (Amaranthaceae). (10) Rosa sericea Wall. Ex Lindl. (Rosaceae). (11) Vitis vinifera L. (Vitaceae). (12) Dieffenbachia amoena Bull. (unresolved, Araceae) (13) Dracaena marginata Hort. (Asparagaceae). (14) Lilium candidum L. (Liliaceae). Technical triplicates. Mean +/− SEM shown. ( g ) The effect of VA extract on luciferase activity in a functional active TGFβ1 reporter cell line. Biological triplicates. Mean +/− SEM shown. Representative of 2 independent assays. ( h ) The levels of TGFβ1 in different fractions eluted from an anion exchange column after loading with indicated factors, using indicated % of 2 M NaCl. Unpaired student’s t-test: * p < 0.05.

Article Snippet: Leaf extract (1 mg/ml in PBS), TGFβ1 antibody (5 µg/ml in PBS; clone 1D11 Bio-Techne), parthenium (1 mg/ml in chloroform; Sigma), artemisinin (1 mg/ml in chloroform; Sigma), full length Pisum sativum L. (Leguminosae) chlorophyll a-b binding protein AB96 (at indicated concentrations; mybiosource TTKKVASSSSPWHGPDGVK YLGPFSGESPSYLTGEFPGDY GWDTAGLSADPETFAK NRELEVIHSRWAMLGAL GCVFPELLSRNGVKFG EAVWFKAGSQIFSEGGL DYLGNPSLVHAQSIL AIWATQVILMGAVEGYRI AGGPLGEVVDPLYPG GS FDPLGLAEVPEAFAE LKVKELKNGRLAMF SMF GFFVPAIVTGKGPLEN LADHLADPVNNN AWSYATNFVPGK), soybean trypsin inhibitor (at indicated concentrations; Sigma) or vehicle (PBS or chloroform) were plated on a high binding 96 well flat-bottomed plate (Greiner) and left at RT overnight.

Techniques: Derivative Assay, Incubation, Neutralization, Luciferase, Activity Assay, Functional Assay

Journal: Scientific Reports

Article Title: Identification of chlorophyll a-b binding protein AB96 as a novel TGFβ1 neutralizing agent

doi: 10.1038/s41598-021-87454-x

Figure Lengend Snippet: Chlorophyll a-b binding protein (CabBP) AB96 binds to and neutralizes active TGFβ1. ( a ) Analyzed mass spectrometry data showing pulled out proteins when using indicated bait proteins when a threshold of min number of peptides = 2, protein threshold = 20% and peptide threshold = 95%. ( b ) The chlorophyll a-b binding protein AB96 peptides from Pisum Sativum (Fabaceae), observed by mass spectrometry are highlighted in yellow. Green represents amino acid modifications: M, oxidation and C, carbamidomethyl. Pink represents Magnesium binding sites. ( c ) Quantitation of chlorophyll a-b binding protein AB96 pulled from VA(aq) using indicated bait protein. ( d ) Concentration of active TGFβ1 eluted (bound fraction) from different concentrations of chlorophyll a-b binding protein AB96 (CabBP) or Soybean trypsin inhibitor (STI) control after incubation for 2 h at RT. Mean +/− SEM shown. Example of 2 independent experiments ( e ) The effect of VA(aq), anti-TGFβ1 antibody, CabBP, STI and relevant controls, on luciferase activity in an active TGFβ1 reporter cell line. Biological quintuplets. Mean +/− SEM shown. Unpaired student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Leaf extract (1 mg/ml in PBS), TGFβ1 antibody (5 µg/ml in PBS; clone 1D11 Bio-Techne), parthenium (1 mg/ml in chloroform; Sigma), artemisinin (1 mg/ml in chloroform; Sigma), full length Pisum sativum L. (Leguminosae) chlorophyll a-b binding protein AB96 (at indicated concentrations; mybiosource TTKKVASSSSPWHGPDGVK YLGPFSGESPSYLTGEFPGDY GWDTAGLSADPETFAK NRELEVIHSRWAMLGAL GCVFPELLSRNGVKFG EAVWFKAGSQIFSEGGL DYLGNPSLVHAQSIL AIWATQVILMGAVEGYRI AGGPLGEVVDPLYPG GS FDPLGLAEVPEAFAE LKVKELKNGRLAMF SMF GFFVPAIVTGKGPLEN LADHLADPVNNN AWSYATNFVPGK), soybean trypsin inhibitor (at indicated concentrations; Sigma) or vehicle (PBS or chloroform) were plated on a high binding 96 well flat-bottomed plate (Greiner) and left at RT overnight.

Techniques: Binding Assay, Mass Spectrometry, Quantitation Assay, Concentration Assay, Incubation, Luciferase, Activity Assay

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Inhibitors of actin polymerization improve monolayer integrity and reverse plasticity. (A–C) LatA (an actin polymerization inhibitor) and U0126 (a MAPK inhibitor) are the only drugs which reduced cellular velocity (A), TEER (B) and correlation length (C) on the CS-exposed epithelia [cells treated with pathway modulators such as LatA, the actin-polymerizing agent JaspA, U0126, the TGFβ1 neutralizer (1D11), or an antagonist of Wnt signaling pathway (DKK1) and compared to vehicle control]. Data is representative of two donors (three to six inserts per donor). (D) None of the inhibitors restored the CS-induced decrease in basal mRNA expression of the epithelial marker CDH1 . (E–J) The levels of the mesenchymal markers ( CDH2 , VIM , SNAI1 , SNAI2 , ZEB1 and ZEB2 ) were significantly reduced relative to GAPDH in CS-exposed epithelia treated with several pathway modulators (LatA, U0126, 1D11 and DKK1) as analyzed by qPCR. (K) LatA and U0126 treatment reduced mean square displacement (MSD) in CS exposed epithelia. Data is representative of three to six inserts from two donors. (L–O) LatA and U0126 treatment improved TEER in CHBE cells (L), LatA and U0126 treatment decreased cellular velocity in COPD epithelia (CHBE) (M), LatA and U0126 treatment reduced correlation length in CHBE cells (N), and LatA and U0126 treatment improved the height of pseudostratified epithelia on COPD epithelia (CHBE) (O). (P) Representative DIC image from three inserts from one donor taken with a 40× oil objective of CHBE treated with or without LatA or U0126 showing improved monolayer height. Scale bars: 25 µm. (Q) LatA and U0126 treatment reduced the MSD with time in CHBE cells approaching levels similar to that of NHBE. Of note, there is not a significant increase in MSD with time in pEMT-induced NHBE. Data is representative of nine inserts, except for cellular velocity and correlation length (six inserts) and height of pseudostratified epithelia (three inserts) from one donor of CHBE. Graphs are shown with median bars. Statistics determined by Kruskal–Wallis test followed by Dunn's multiple comparison test, with P <0.05 considered statistically significant.

Article Snippet: The NHBE cells at the ALI were exposed to CS for 10 days and simultaneously basolaterally treated with an antagonist of Wnt signaling pathway (recombinant human DKK1 protein, 50 ng/ml, R&D Systems), TGFβ1 neutralizer [TGFβ-1,2,3 monoclonal antibody (1D11), 1.25 μg/ml, Thermo Fisher Scientific], MAPK inhibitor (U0126, 15 μM, Cell Signaling Technology ® ), an inhibitor of actin polymerization (Latrunculin A, 0.625 μM, Sigma-Aldrich) or the actin filament-polymerizing and stabilizing agent (Jasplakinolide A, 0.25 μM, Sigma-Aldrich).

Techniques: Control, Expressing, Marker, Comparison

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 inhibited PI3K/AKT and Raf/MEK/ERK signaling pathways triggered by TGFβ1. (A) The total proteins extracted from cells were analyzed for the expression of AKT, ERK and Bax using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-Bax and anti-GAPDH. (B) ELISA analysis of TGFβ1 secretion from AsPC-1/vector and AsPC-1/TIPE2 cells. (C) Immunohistochemistry analysis of the TGFβ1 expression in AsPC-1/vector and AsPC-1/TIPE2 tumor tissues. (D) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in AsPC-1/vector and AsPC-1/TIPE2 cells. (E) AsPC-1 cells were seeded in 6-well plate and added with or without anti-TGFβ1 antibody or rhTGFβ1 protein. After 48 h incubation, the total proteins extracted from the cultured AsPC-1 cells were analyzed for the expression of AKT and ERK using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK and anti-GAPDH. Data shown were representative of three independent experiments. Values are presented as means ± SD. ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Incubation, Cell Culture

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 suppressed the growth of pancreatic cancer through inhibiting TGFβ1 expression in subcutaneous tumor model. (A) The tumor volume was measured weekly one week after tumor inoculation and the tumor growth curve was calculated. (B, C) The tumors were isolated, weighted and photographed five weeks after tumor cells inoculation. (D) ELISA analysis of TGFβ1 secretion from Panc02/vector and Panc02/TIPE2 cells. (E) Immunohistochemistry analysis of the TGFβ1 expression in Panc02/vector and Panc02/TIPE2 tumor tissues. (F) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in Panc02/vector and Panc02/TIPE2 cells. The data shown are the representative of three experiments. Values are presented as means ± SD. *p < 0.05, ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Western Blot

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: Mφ from S. japonicum-infected mice are sufficient to induce Tregs and exhibit a tolerogenic phenotype characterized by expressing high levels of TGF-β and IL-10. (a, b) Purified CD4+ T cells from normal mice were co-cultured with Mφ from normal or S. japonicum-infected mice for 3 days. CD4+CD25+Foxp3+ Tregs were analyzed by FCM and cells were gated on CD4+ T cells. (c, d) At 13 weeks post-infection, single-cell suspensions of splenocytes or liver cells from S. japonicum-infected mice were stimulated with phorbol myristate acetate and ionomycin in the presence of Golgistop for 6 h. Cells were surface stained with anti-CD11b and anti-F4/80 then intracellularly stained with antibodies against TNF-α, IL-4, IL-6, IL-10, TGF-β1, or isotype antibody and analyzed by FCM. Cells were gated on CD11b+F4/80+ Mφ. FCM plots are representative of three independent experiments. Data are means ± s.d. of triplicate cultures or 5 mice and representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Infection, Expressing, Purification, Cell Culture, Staining

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-treated Mφ display a phenotype resembling that of tolerogenic Mφ characterized by expressing high levels of TGF-β and IL-10. (a) Purified Mφ from normal mice were cultured for 2 days in medium alone or in the presence of 0.1 μg mL−1 SjHSP60 or 1 μg mL−1 LPS. The mRNA expression of cytokines in Mφ were determined by qPCR and expressed as fold increases over control. (b) ELISA quantification of cytokines in supernatants of Mφ cultures. Data are means ± s.d. of triplicate cultures and representative of three independent experiments. **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Expressing, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: Production of TGF-β by SjHSP60-Mφ is necessary for conversion of CD4+CD25− T cells into Tregs. (a) At 0, 3, 5, 8 and 13 weeks post-infection, serum TGF-β levels were measured by ELISA. (b) ELISA measurements of TGF-β in serum samples of mice injected with SjHSP60 or PBS. (c) ELISA measurements of cytokines in supernatants of purified CD4+CD25− T cells cultured for 3 days in medium alone or with Mφ that had been unstimulated or exposed to SjHSP60 or LPS for 2 days. (d) Mφ were prestimulated with 0.1 μg mL−1 SjHSP60 or medium for 2 days. Purified CD4+CD25− T cells were cultured for 3 days with medium, SjHSP60, or prestimulated Mφ above. Co-cultures were set up with or without SB-431542 (20 μmol L−1) or neutralizing TGF-β1 antibody (5 μg mL−1). Frequency of CD4+CD25+Foxp3+ Tregs was determined by FCM. FCM plots are representative of three independent experiments with similar results and cells were gated on CD4+ T cells. (e) The bar graph indicates average percentages ± s.d. of Tregs. Data are expressed as means ± s.d. of 6 mice or triplicate cultures and representative of two or three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Injection, Purification, Cell Culture

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-triggered TGF-β production in Mφ is TLR4-dependent. (a, b) Purified Mφ from TLR2−/−, TLR4−/− mice or control WT littermates were stimulated with or without 0.1 μg mL−1 SjHSP60 for 2 days. In some cultures, purified Mφ from control littermates were pretreated with medium alone, or with 20 μg mL−1 of anti-TLR2, anti-TLR4 blocking or isotype-matched antibodies for 1 h at 4°C. After stimulation, TGF-β production was detected by ELISA. Data are means ± s.d. of triplicate cultures and representative of three independent experiments. **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Purification, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-triggered TGF-β production and Treg conversion are MyD88-independent. (a, b) MyD88+/+ or MyD88−/− Mφ were stimulated with 0.1 μg mL−1 SjHSP60 for 2 days. TGF-β mRNA expression was detected by qPCR and expressed as fold increases over unstimulated MyD88+/+ Mφ (a). Culture supernatants were collected for determining TGF-β production by ELISA (b). (c, d) Purified CD4+CD25− T cells were co-cultured with MyD88+/+ or MyD88−/− Mφ prestimulated with 0.1 μg mL−1 SjHSP60 or medium. After 3 days, percentages of CD4+CD25+Foxp3+ Tregs were analyzed by FCM. FCM plots are representative of three independent experiments with consistent results and cells were gated on CD4+ T cells. The bar graph indicates average percentages ± s.d. of Tregs. Data are means ± s.d. of triplicate cultures and representative of two or three independent experiments. **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-triggered TGF-β production and Treg conversion are Mal-dependent. (a) Purified Mφ from MyD88−/− mice or control WT littermates were cultured with or without 0.1 μg mL−1 SjHSP60 for 2 days. Mal mRNA levels were determined by qPCR and expressed as fold increases over unstimulated MyD88+/+ Mφ. (b, c) Effect of specific siRNA on endogenous Mal expression in RAW264.7 cells was analyzed by qPCR (b) and Western blot (c). (d, e) RAW264.7 cells transfected with Mal siRNA or control siRNA were stimulated with 0.1 μg mL−1 of SjHSP60. After 2 days, TGF-β levels in supernatants were detected by ELISA (e), while cells were washed and co-cultured with purified CD4+CD25− T cells for three additional days. Percentages of CD4+CD25+Foxp3+ Tregs in co-cultures were measured by FCM and cells were gated on CD4+ T cells (e). (f) The bar graph indicates average percentages ± s.d. of Tregs. Western blots and FCM plots are representative of three independent experiments with consistent results. Data are means ± s.d. of triplicate cultures and representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Purification, Cell Culture, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Cell Biology

Article Title: A “traffic control” role for TGFβ3: orchestrating dermal and epidermal cell motility during wound healing

doi: 10.1083/jcb.200507111

Figure Lengend Snippet: TGFβ3 but not TGFβ1 or TGFβ2 in human serum selectively blocks dermal cell migration. DFs, HDMECs, and HKs were serum starved overnight and subjected to migration assays on collagen in the absence or presence of plasma, serum, or serum with added increasing amounts of anti-TGFβ neutralizing antibodies. The four antibodies include a pan-antibody against all three TGFβ isoforms and three other antibodies specifically against the individual TGFβ1, TGFβ2, or TGFβ3. The amounts of these antibodies were predetermined to neutralize the same amounts of their corresponding TGFβ isoforms. (A) Anti-TGFβ3 antibody released the inhibition of serum in DF migration, whereas anti-TGFβ1 and anti-TGFβ2 antibodies did not. This experiment was repeated four times. Error bars represent SEM. (B) The addition of anti-TGFβ3 antibody to serum converted to a promotility agent for both DFs and HDMECs. The cells were subjected to in vitro wound-healing assays under either serum-free, serum, or serum with added anti-TGFβ3 neutralizing antibodies. After 16 h, the wound closures were photographed, and the AGs were quantitated as described previously . AGs are indicated by lines and arrows; some gaps were too close to insert an arrow. The results shown here were reproducible in two independent experiments.

Article Snippet: Anti–human TGFβ (1, 2, and 3) pan-neutralizing antibody and neutralizing antibodies specifically against human TGFβ1, TGFβ2, or TGFβ3 were obtained from R&D Systems.

Techniques: Migration, Clinical Proteomics, Inhibition, In Vitro

Journal: The Journal of Cell Biology

Article Title: A “traffic control” role for TGFβ3: orchestrating dermal and epidermal cell motility during wound healing

doi: 10.1083/jcb.200507111

Figure Lengend Snippet: In vitro and in vivo human skin profiles of TβRs (I, II, and III) and their transmission of TGFβ-stimulated Smad 2/3 activation. (A) Equal amounts of cell lysates of serum-starved HKs, MCs, DFs, and HDMECs were resolved in three SDS gels and subjected to Western blot analyses with antibodies against TβRI/ALK5 (a), TβRII (c), or TβRIII/betaglycan (e). Each lower part of the three membranes was blotted with an anti–β-actin antibody as a control (b, d, and f). The expression was quantitated by a densitometry scan, giving rise to a ratio of TβR band intensity over its corresponding β-actin band intensity. (B) Normal human skin sections were subjected to indirect immunofluorescence staining with the antibodies against TβRI (a), TβRII (f), TβRIII (k), DAPI (b, g, and l), rhodamine-conjugated phalloidin (c, h, and m), or corresponding IgG controls (see Materials and methods). The images show skin tissue distribution of the TβRs (a, f, and k), nuclei (b, g, and l), F-actin (c, h, and m), and all three stains merged (d, i, and n). A section of the anti-TβRI antibody staining (a, dotted box) was enlarged in the inset to visualize cellular levels of staining. Bar, 20 μm. (C) The cells were either untreated or treated with the indicated concentrations of TGFβ3 for 15 min, and equalized cell lysates (40 μg/lane) were subjected to Western blot analyses with either antiphospho-Smad 2/3 (a, c, and e) or anti-Smad 2/3 (b, d, and f) antibodies. (D) The cells were either untreated or treated with 2.5 ng/ml TGFβ3 for the indicated periods of time. Equalized cell lysates were subjected to Western blot analyses with either antiphospho-Smad 2/3 (a, c, and e) or anti-Smad 2/3 (b, d, and f) antibodies.

Article Snippet: Anti–human TGFβ (1, 2, and 3) pan-neutralizing antibody and neutralizing antibodies specifically against human TGFβ1, TGFβ2, or TGFβ3 were obtained from R&D Systems.

Techniques: In Vitro, In Vivo, Transmission Assay, Activation Assay, Western Blot, Control, Expressing, Immunofluorescence, Staining