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recombinant human latent tgf β1  (R&D Systems)


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    R&D Systems recombinant human latent tgf β1
    <t>TGF-β1</t> <t>upregulates</t> intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Recombinant Human Latent Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+latent+tgf+%CE%B21/Recombinant+Human+Latent+TGF-beta+1+Protein/pmc12984908-94-2-7
    Average 94 stars, based on 42 article reviews
    recombinant human latent tgf β1 - by Bioz Stars, 2026-09
    94/100 stars

    Images

    1) Product Images from "Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop"

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    Journal: Cells

    doi: 10.3390/cells15050404

    TGF-β1 upregulates intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Figure Legend Snippet: TGF-β1 upregulates intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).

    Techniques Used: Western Blot, Staining, Control, Immunofluorescence, Fluorescence

    TGF-β1 upregulation of NEU3 occurs in the presence of a transcription inhibitor. ( A ) Human lung fibroblasts were pre-incubated with either 1 µg/mL of the transcription inhibitor actinomycin D (Act D) or DMSO buffer control for 10 min. Cells were then washed and exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for caldesmon or NEU3 using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of two male and two female cell lines. Bars are 50 µm. Yellow arrows are examples of staining artifacts that were not included in fluorescence quantification. ( B , C ) Quantification of immunofluorescence. Actinomycin D-treated cells are shown in orange and control cells are shown in black, circles indicate healthy cell donors and triangles indicate cells from IPF patients. ( D ) Human lung fibroblasts were pre-incubated with either 50 µg/mL of the translation inhibitor cycloheximide or buffer control for 10 min. Cells were then treated with TGF-β1 and stained for NEU3 by immunofluorescence. Bars are 50 µm. ( E ) Quantification of immunofluorescence. Cycloheximide treated cells are shown in blue and control treated cells are shown in black. Circles indicate healthy cell donors and triangles indicate cells from IPF patients. Values are normalized to the 0 min control. Bars are mean ± SEM, n = 4 (two male and two female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). # p < 0.05 ## p < 0.01 ### p < 0.001 (Unpaired t -test comparing actinomycin D or cycloheximide to control for each time point).
    Figure Legend Snippet: TGF-β1 upregulation of NEU3 occurs in the presence of a transcription inhibitor. ( A ) Human lung fibroblasts were pre-incubated with either 1 µg/mL of the transcription inhibitor actinomycin D (Act D) or DMSO buffer control for 10 min. Cells were then washed and exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for caldesmon or NEU3 using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of two male and two female cell lines. Bars are 50 µm. Yellow arrows are examples of staining artifacts that were not included in fluorescence quantification. ( B , C ) Quantification of immunofluorescence. Actinomycin D-treated cells are shown in orange and control cells are shown in black, circles indicate healthy cell donors and triangles indicate cells from IPF patients. ( D ) Human lung fibroblasts were pre-incubated with either 50 µg/mL of the translation inhibitor cycloheximide or buffer control for 10 min. Cells were then treated with TGF-β1 and stained for NEU3 by immunofluorescence. Bars are 50 µm. ( E ) Quantification of immunofluorescence. Cycloheximide treated cells are shown in blue and control treated cells are shown in black. Circles indicate healthy cell donors and triangles indicate cells from IPF patients. Values are normalized to the 0 min control. Bars are mean ± SEM, n = 4 (two male and two female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). # p < 0.05 ## p < 0.01 ### p < 0.001 (Unpaired t -test comparing actinomycin D or cycloheximide to control for each time point).

    Techniques Used: Incubation, Control, Staining, Immunofluorescence, Fluorescence

    NEU3 functions in a positive feedback loop with TGF-β1 that is blocked by NEU3 inhibitors. ( A ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5 (3 male and 2 female cells lines). * p < 0.05 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control). $ p < 0.05, $$ p < 0.01 (Unpaired t -test comparing male to female). ( B ) Human lung fibroblast cells were exposed to 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue respectively) and the indicated concentrations of latent TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. ( C ) Human lung fibroblasts were exposed to 0 or 0.3 ng/mL of TGF-β1 (indicated with gray and green respectively) and the indicated concentrations of the retentate of latent TGF-β1 (purified with a 50 kDa spin filter) for 5 min, fixed, and stained with NEU3 using immunofluorescence. ( D ) Human lung fibroblast cells were exposed to 0.1 ng/mL of latent TGF-β1 (L-TGF-β1) with either 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue fill respectively, white fill indicates no latent TGF-β1 or added TGF-β1) in the presence or absence of NEU3 inhibitors: 2-AP and AMPCA (indicated with stripes and dots respectively). Cells were fixed and stained for NEU3 using immunofluorescence. For ( B – D ), values are normalized to media control. For ( B , D ) bars are mean ± SEM, n = 3 (2 male and 1 female cell line) for ( C ) bars are mean ± SEM, n = 4 (2 male and 2 female cell lines). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.001 (One-way ANOVA, Dunnet’s test compared to media control). # p < 0.05, ## p < 0.01, ### p < 0.001 (Unpaired t -test comparing with and without inhibitor for each concentration of TGF-β1).
    Figure Legend Snippet: NEU3 functions in a positive feedback loop with TGF-β1 that is blocked by NEU3 inhibitors. ( A ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5 (3 male and 2 female cells lines). * p < 0.05 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control). $ p < 0.05, $$ p < 0.01 (Unpaired t -test comparing male to female). ( B ) Human lung fibroblast cells were exposed to 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue respectively) and the indicated concentrations of latent TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. ( C ) Human lung fibroblasts were exposed to 0 or 0.3 ng/mL of TGF-β1 (indicated with gray and green respectively) and the indicated concentrations of the retentate of latent TGF-β1 (purified with a 50 kDa spin filter) for 5 min, fixed, and stained with NEU3 using immunofluorescence. ( D ) Human lung fibroblast cells were exposed to 0.1 ng/mL of latent TGF-β1 (L-TGF-β1) with either 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue fill respectively, white fill indicates no latent TGF-β1 or added TGF-β1) in the presence or absence of NEU3 inhibitors: 2-AP and AMPCA (indicated with stripes and dots respectively). Cells were fixed and stained for NEU3 using immunofluorescence. For ( B – D ), values are normalized to media control. For ( B , D ) bars are mean ± SEM, n = 3 (2 male and 1 female cell line) for ( C ) bars are mean ± SEM, n = 4 (2 male and 2 female cell lines). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.001 (One-way ANOVA, Dunnet’s test compared to media control). # p < 0.05, ## p < 0.01, ### p < 0.001 (Unpaired t -test comparing with and without inhibitor for each concentration of TGF-β1).

    Techniques Used: Staining, Immunofluorescence, Control, Purification, Concentration Assay

    Latency-associated peptide upregulates NEU3 at 5 min and functions synergistically with TGF-β1. Human lung fibroblast cells were exposed to the indicated concentrations of ( A ) LAP alone and ( B ) suboptimal concentrations of LAP, TGF-β1, and a combination for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5–11 (4–6 male and 1–3 female cells lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control).
    Figure Legend Snippet: Latency-associated peptide upregulates NEU3 at 5 min and functions synergistically with TGF-β1. Human lung fibroblast cells were exposed to the indicated concentrations of ( A ) LAP alone and ( B ) suboptimal concentrations of LAP, TGF-β1, and a combination for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5–11 (4–6 male and 1–3 female cells lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control).

    Techniques Used: Staining, Immunofluorescence, Control

    DDX3 mediates NEU3 upregulation by TGF-β1 and LAP. Human lung fibroblasts were pre-incubated with either 10 µM of RK-33 or an equal volume dilution of DMSO in protein free medium for 10 min. Protein free medium with or without ( A ) 10 ng/mL TGF-β1 or ( B ) 0.3 ng/mL LAP was added to wells for 5 min. Cells were then washed, fixed, and stained for NEU3 using immunofluorescence. RK-33 treated cells are indicated with red bars. Values are normalized to 0 min controls. Bars are mean ± SEM, n = 3–4 (1–2 male and 2 female cells lines). ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control). Human lung fibroblasts were treated with ( C – E ) 10 ng/mL of recombinant human active TGF-β1 or ( F – H ) 0.3 ng/mL of recombinant human LAP for the indicated times. m indicates minutes, h hour. The 0 min samples were treated with protein free media only. Phosphorylated proteins were isolated using TALON PMAC magnetic beads. Phosphorylated proteins and supernatants of total cell lysates were stained for DDX3 using Western blots. Blots are representative of five donors (four male and one female). ( D , E , G , H ) Quantification of Western blots. Band densities of phosphorylated and total DDX3 were normalized to the integrated density of silver- or Coomassie-stained gels respectively of total protein for each sample , then values were normalized to the 0 min control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM, n = 5 (4 male and 1 female). * p < 0.05, ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control).
    Figure Legend Snippet: DDX3 mediates NEU3 upregulation by TGF-β1 and LAP. Human lung fibroblasts were pre-incubated with either 10 µM of RK-33 or an equal volume dilution of DMSO in protein free medium for 10 min. Protein free medium with or without ( A ) 10 ng/mL TGF-β1 or ( B ) 0.3 ng/mL LAP was added to wells for 5 min. Cells were then washed, fixed, and stained for NEU3 using immunofluorescence. RK-33 treated cells are indicated with red bars. Values are normalized to 0 min controls. Bars are mean ± SEM, n = 3–4 (1–2 male and 2 female cells lines). ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control). Human lung fibroblasts were treated with ( C – E ) 10 ng/mL of recombinant human active TGF-β1 or ( F – H ) 0.3 ng/mL of recombinant human LAP for the indicated times. m indicates minutes, h hour. The 0 min samples were treated with protein free media only. Phosphorylated proteins were isolated using TALON PMAC magnetic beads. Phosphorylated proteins and supernatants of total cell lysates were stained for DDX3 using Western blots. Blots are representative of five donors (four male and one female). ( D , E , G , H ) Quantification of Western blots. Band densities of phosphorylated and total DDX3 were normalized to the integrated density of silver- or Coomassie-stained gels respectively of total protein for each sample , then values were normalized to the 0 min control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM, n = 5 (4 male and 1 female). * p < 0.05, ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control).

    Techniques Used: Incubation, Staining, Immunofluorescence, Control, Recombinant, Isolation, Magnetic Beads, Western Blot

    Proposed model of rapid NEU3 upregulation. Active TGF-β1 and LAP increase DDX3 interaction with NEU3 mRNA either by increasing binding to the common motif in NEU3 mRNA, found in 179 other proteins that are translationally regulated by TGF-β1 (orange), or to G-quadruplex structures (green; the image labels 4 of the 32 predicted G-quadruplex structures). DDX3 stimulates rapid NEU3 translation. Once translated and released from cells, NEU3 activates latent TGF-β1 (L-TGF-β1), releasing active TGF-β1 from its complex with LAP. TGF-β1 and LAP synergistically upregulate NEU3 translation leading to a positive feedback loop. DDX3 is inhibited by RK-33, and NEU3 is inhibited by 2-AP and AMPCA, all three of which can disrupt this loop and the rapid 5 min upregulation of NEU3. This figure was created using BioRender elements ( https://biorender.com/ (accessed on 16 July 2025)); structures of NEU3 and TGF-β1 were predicted using AlphaFold 3 .
    Figure Legend Snippet: Proposed model of rapid NEU3 upregulation. Active TGF-β1 and LAP increase DDX3 interaction with NEU3 mRNA either by increasing binding to the common motif in NEU3 mRNA, found in 179 other proteins that are translationally regulated by TGF-β1 (orange), or to G-quadruplex structures (green; the image labels 4 of the 32 predicted G-quadruplex structures). DDX3 stimulates rapid NEU3 translation. Once translated and released from cells, NEU3 activates latent TGF-β1 (L-TGF-β1), releasing active TGF-β1 from its complex with LAP. TGF-β1 and LAP synergistically upregulate NEU3 translation leading to a positive feedback loop. DDX3 is inhibited by RK-33, and NEU3 is inhibited by 2-AP and AMPCA, all three of which can disrupt this loop and the rapid 5 min upregulation of NEU3. This figure was created using BioRender elements ( https://biorender.com/ (accessed on 16 July 2025)); structures of NEU3 and TGF-β1 were predicted using AlphaFold 3 .

    Techniques Used: Binding Assay

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    Recombinant:

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    Article Snippet: .. Recombinant human LAP and human latent TGF-β1, obtained from R&D Systems, Inc (Minneapolis, MN, USA [catalog no. 246-LP, carrier-free and 299-LT, respectively]), were coupled via a standard amine-coupling procedure to the flow cells of a CM5 sensor chip until a level of 1000 resonance units (RU) was reached. ..

    Article Title: High fructose consumption aggravates inflammation by promoting effector T cell generation via inducing metabolic reprogramming
    Article Snippet: For Th17 cell induction, T cells were induced with recombinant human TGF-β1 (2 ng/mL, PEPROTECH) plus recombinant mouse IL-6 (50 ng/mL, PEPROTECH). .. For TGF-β activation experiments, T cells were cultured with recombinant mouse IL-6, with or without recombinant human latent TGF-β1 (10 ng/mL, R&D Systems). .. TGF-β signaling was blocked using anti-TGF-β (50 μg/mL, Bio X Cell) and SB431542 (5 μM, Selleck.cn).

    Activation Assay:

    Article Title: High fructose consumption aggravates inflammation by promoting effector T cell generation via inducing metabolic reprogramming
    Article Snippet: For Th17 cell induction, T cells were induced with recombinant human TGF-β1 (2 ng/mL, PEPROTECH) plus recombinant mouse IL-6 (50 ng/mL, PEPROTECH). .. For TGF-β activation experiments, T cells were cultured with recombinant mouse IL-6, with or without recombinant human latent TGF-β1 (10 ng/mL, R&D Systems). .. TGF-β signaling was blocked using anti-TGF-β (50 μg/mL, Bio X Cell) and SB431542 (5 μM, Selleck.cn).

    Cell Culture:

    Article Title: High fructose consumption aggravates inflammation by promoting effector T cell generation via inducing metabolic reprogramming
    Article Snippet: For Th17 cell induction, T cells were induced with recombinant human TGF-β1 (2 ng/mL, PEPROTECH) plus recombinant mouse IL-6 (50 ng/mL, PEPROTECH). .. For TGF-β activation experiments, T cells were cultured with recombinant mouse IL-6, with or without recombinant human latent TGF-β1 (10 ng/mL, R&D Systems). .. TGF-β signaling was blocked using anti-TGF-β (50 μg/mL, Bio X Cell) and SB431542 (5 μM, Selleck.cn).

    Concentration Assay:

    Article Title: Anti-TGF-β1 aptamer enhances therapeutic effect of tyrosine kinase inhibitor, gefitinib, on non-small cell lung cancer in xenograft model
    Article Snippet: .. After immobilization of aptamer, the following proteins at final concentration of 500 nM were injected for 120 s: human TGF-β1 (PeproTech), mouse TGF-β1 (R&D systems, MN, USA), human TGF-β2 (PeproTech), human TGF-β3 (PeproTech), human latent TGF-β1 (R&D system), and human alpha-Thrombin (Haematologic Technologies, VT, USA) as a negative control. ..

    Injection:

    Article Title: Anti-TGF-β1 aptamer enhances therapeutic effect of tyrosine kinase inhibitor, gefitinib, on non-small cell lung cancer in xenograft model
    Article Snippet: .. After immobilization of aptamer, the following proteins at final concentration of 500 nM were injected for 120 s: human TGF-β1 (PeproTech), mouse TGF-β1 (R&D systems, MN, USA), human TGF-β2 (PeproTech), human TGF-β3 (PeproTech), human latent TGF-β1 (R&D system), and human alpha-Thrombin (Haematologic Technologies, VT, USA) as a negative control. ..

    Negative Control:

    Article Title: Anti-TGF-β1 aptamer enhances therapeutic effect of tyrosine kinase inhibitor, gefitinib, on non-small cell lung cancer in xenograft model
    Article Snippet: .. After immobilization of aptamer, the following proteins at final concentration of 500 nM were injected for 120 s: human TGF-β1 (PeproTech), mouse TGF-β1 (R&D systems, MN, USA), human TGF-β2 (PeproTech), human TGF-β3 (PeproTech), human latent TGF-β1 (R&D system), and human alpha-Thrombin (Haematologic Technologies, VT, USA) as a negative control. ..



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    <t>TGF-β1</t> <t>upregulates</t> intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Recombinant Human Latent Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+latent+tgf+%CE%B21/Recombinant+Human+Latent+TGF-beta+1+Protein/pmc12984908-94-2-7
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    human recombinant ltgf β1  (R&D Systems)
    93
    R&D Systems human recombinant ltgf β1
    <t>TGF-β1</t> <t>upregulates</t> intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Human Recombinant Ltgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+latent+tgf+%CE%B21/Recombinant+Human+Latent+TGF-beta+1+Protein%2C+CF/pm41274617-70-0-8
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    human latent tgf β1  (R&D Systems)
    94
    R&D Systems human latent tgf β1
    <t>TGF-β1</t> <t>upregulates</t> intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Human Latent Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+latent+tgf+%CE%B21/Recombinant+Human+Latent+TGF-beta+1+Protein/pmc12379281-306-16-21
    Average 94 stars, based on 1 article reviews
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    latent tgf β1  (R&D Systems)
    93
    R&D Systems latent tgf β1
    <t>TGF-β1</t> <t>upregulates</t> intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Latent Tgf β1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+latent+tgf+%CE%B21/Recombinant+Human+Latent+TGF-beta+1+Protein%2C+CF/pmc12168093-54-12-14
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    tgf β1  (MedChemExpress)
    93
    MedChemExpress tgf β1
    <t>TGF-β1</t> <t>upregulates</t> intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).
    Tgf β1, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TGF-β1 upregulates intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).

    Journal: Cells

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    doi: 10.3390/cells15050404

    Figure Lengend Snippet: TGF-β1 upregulates intracellular and extracellular NEU3 levels within minutes. Human lung fibroblast cells were exposed to 10 ng/mL of TGF-β1 for the indicated times, lysed, and Western blots were stained for intracellular ( A ) NEU3 or ( B ) caldesmon. m indicates minutes, h hours. The 0 min samples were treated with protein free media only. Images are representative of three male and two female cell lines, each from a different donor. Molecular weights in kDa are indicated on the left. ( C , D ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample . For NEU3, the 55 and 48 kDa bands were added together. For each cell line, three or more assays were done, and in each assay, after normalizing to Coomassie densities, values were normalized to the protein free media control at that time point . The results were then averaged for each cell line. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (three male and two female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 min control). $ p < 0.05 (Unpaired t -test comparing male to female). ( E ) Human lung fibroblasts were exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for intracellular NEU3 (rows 1 and 2) or caldesmon (rows 3 and 4) using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of four male and three female cell lines, each from a different donor. Rows 2 and 4 show magnified views of images from rows 1 and 3 respectively. Bar is 100 µm for rows 1 and 3 and 50 µm for rows 2 and 4. Yellow arrows are examples of artifacts, detached cells, or abnormal appearing cells that were not included in fluorescence quantification. ( F , G ) Quantification of immunofluorescence, m indicates minutes, h hours. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to protein free media control for each time point . Bars are mean ± SEM, n ≥ 7 (4 male and 3 female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). ( H ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min. After pipetting up and down gently, liquid was collected off cells, and stained for NEU3 on Western blots. Molecular weights in kDa are indicated on the left. ( I ) Quantification of Western blots. Band densities were normalized to the integrated density of Coomassie-stained gels of total protein for each sample, then values were normalized to the 0 ng/mL control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM of the five averages (two male and three female cell lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnett’s test compared to the 0 ng/mL control).

    Article Snippet: Where indicated, recombinant human latent TGF-β1 (299-LT/CF, R&D systems), recombinant human latency associated peptide (LAP-H5213, Acro Biosystems, Newark, DE, USA), and lipopolysaccharide (437627, Caliobiochem Corporation, La Jolla, CA, USA) were diluted in protein free medium and added to cells.

    Techniques: Western Blot, Staining, Control, Immunofluorescence, Fluorescence

    TGF-β1 upregulation of NEU3 occurs in the presence of a transcription inhibitor. ( A ) Human lung fibroblasts were pre-incubated with either 1 µg/mL of the transcription inhibitor actinomycin D (Act D) or DMSO buffer control for 10 min. Cells were then washed and exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for caldesmon or NEU3 using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of two male and two female cell lines. Bars are 50 µm. Yellow arrows are examples of staining artifacts that were not included in fluorescence quantification. ( B , C ) Quantification of immunofluorescence. Actinomycin D-treated cells are shown in orange and control cells are shown in black, circles indicate healthy cell donors and triangles indicate cells from IPF patients. ( D ) Human lung fibroblasts were pre-incubated with either 50 µg/mL of the translation inhibitor cycloheximide or buffer control for 10 min. Cells were then treated with TGF-β1 and stained for NEU3 by immunofluorescence. Bars are 50 µm. ( E ) Quantification of immunofluorescence. Cycloheximide treated cells are shown in blue and control treated cells are shown in black. Circles indicate healthy cell donors and triangles indicate cells from IPF patients. Values are normalized to the 0 min control. Bars are mean ± SEM, n = 4 (two male and two female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). # p < 0.05 ## p < 0.01 ### p < 0.001 (Unpaired t -test comparing actinomycin D or cycloheximide to control for each time point).

    Journal: Cells

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    doi: 10.3390/cells15050404

    Figure Lengend Snippet: TGF-β1 upregulation of NEU3 occurs in the presence of a transcription inhibitor. ( A ) Human lung fibroblasts were pre-incubated with either 1 µg/mL of the transcription inhibitor actinomycin D (Act D) or DMSO buffer control for 10 min. Cells were then washed and exposed to 10 ng/mL of TGF-β1 for the indicated times, fixed, and stained for caldesmon or NEU3 using immunofluorescence. The 0 min samples were treated with protein free media only. Images are representative of two male and two female cell lines. Bars are 50 µm. Yellow arrows are examples of staining artifacts that were not included in fluorescence quantification. ( B , C ) Quantification of immunofluorescence. Actinomycin D-treated cells are shown in orange and control cells are shown in black, circles indicate healthy cell donors and triangles indicate cells from IPF patients. ( D ) Human lung fibroblasts were pre-incubated with either 50 µg/mL of the translation inhibitor cycloheximide or buffer control for 10 min. Cells were then treated with TGF-β1 and stained for NEU3 by immunofluorescence. Bars are 50 µm. ( E ) Quantification of immunofluorescence. Cycloheximide treated cells are shown in blue and control treated cells are shown in black. Circles indicate healthy cell donors and triangles indicate cells from IPF patients. Values are normalized to the 0 min control. Bars are mean ± SEM, n = 4 (two male and two female cell lines). * p < 0.05 ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to the 0 min control). # p < 0.05 ## p < 0.01 ### p < 0.001 (Unpaired t -test comparing actinomycin D or cycloheximide to control for each time point).

    Article Snippet: Where indicated, recombinant human latent TGF-β1 (299-LT/CF, R&D systems), recombinant human latency associated peptide (LAP-H5213, Acro Biosystems, Newark, DE, USA), and lipopolysaccharide (437627, Caliobiochem Corporation, La Jolla, CA, USA) were diluted in protein free medium and added to cells.

    Techniques: Incubation, Control, Staining, Immunofluorescence, Fluorescence

    NEU3 functions in a positive feedback loop with TGF-β1 that is blocked by NEU3 inhibitors. ( A ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5 (3 male and 2 female cells lines). * p < 0.05 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control). $ p < 0.05, $$ p < 0.01 (Unpaired t -test comparing male to female). ( B ) Human lung fibroblast cells were exposed to 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue respectively) and the indicated concentrations of latent TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. ( C ) Human lung fibroblasts were exposed to 0 or 0.3 ng/mL of TGF-β1 (indicated with gray and green respectively) and the indicated concentrations of the retentate of latent TGF-β1 (purified with a 50 kDa spin filter) for 5 min, fixed, and stained with NEU3 using immunofluorescence. ( D ) Human lung fibroblast cells were exposed to 0.1 ng/mL of latent TGF-β1 (L-TGF-β1) with either 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue fill respectively, white fill indicates no latent TGF-β1 or added TGF-β1) in the presence or absence of NEU3 inhibitors: 2-AP and AMPCA (indicated with stripes and dots respectively). Cells were fixed and stained for NEU3 using immunofluorescence. For ( B – D ), values are normalized to media control. For ( B , D ) bars are mean ± SEM, n = 3 (2 male and 1 female cell line) for ( C ) bars are mean ± SEM, n = 4 (2 male and 2 female cell lines). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.001 (One-way ANOVA, Dunnet’s test compared to media control). # p < 0.05, ## p < 0.01, ### p < 0.001 (Unpaired t -test comparing with and without inhibitor for each concentration of TGF-β1).

    Journal: Cells

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    doi: 10.3390/cells15050404

    Figure Lengend Snippet: NEU3 functions in a positive feedback loop with TGF-β1 that is blocked by NEU3 inhibitors. ( A ) Human lung fibroblast cells were exposed to the indicated concentrations of TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5 (3 male and 2 female cells lines). * p < 0.05 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control). $ p < 0.05, $$ p < 0.01 (Unpaired t -test comparing male to female). ( B ) Human lung fibroblast cells were exposed to 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue respectively) and the indicated concentrations of latent TGF-β1 for 5 min, fixed, and stained for NEU3 using immunofluorescence. ( C ) Human lung fibroblasts were exposed to 0 or 0.3 ng/mL of TGF-β1 (indicated with gray and green respectively) and the indicated concentrations of the retentate of latent TGF-β1 (purified with a 50 kDa spin filter) for 5 min, fixed, and stained with NEU3 using immunofluorescence. ( D ) Human lung fibroblast cells were exposed to 0.1 ng/mL of latent TGF-β1 (L-TGF-β1) with either 0, 0.1, 0.3 or 1 ng/mL of TGF-β1 (indicated with gray, orange, green, and blue fill respectively, white fill indicates no latent TGF-β1 or added TGF-β1) in the presence or absence of NEU3 inhibitors: 2-AP and AMPCA (indicated with stripes and dots respectively). Cells were fixed and stained for NEU3 using immunofluorescence. For ( B – D ), values are normalized to media control. For ( B , D ) bars are mean ± SEM, n = 3 (2 male and 1 female cell line) for ( C ) bars are mean ± SEM, n = 4 (2 male and 2 female cell lines). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.001 (One-way ANOVA, Dunnet’s test compared to media control). # p < 0.05, ## p < 0.01, ### p < 0.001 (Unpaired t -test comparing with and without inhibitor for each concentration of TGF-β1).

    Article Snippet: Where indicated, recombinant human latent TGF-β1 (299-LT/CF, R&D systems), recombinant human latency associated peptide (LAP-H5213, Acro Biosystems, Newark, DE, USA), and lipopolysaccharide (437627, Caliobiochem Corporation, La Jolla, CA, USA) were diluted in protein free medium and added to cells.

    Techniques: Staining, Immunofluorescence, Control, Purification, Concentration Assay

    Latency-associated peptide upregulates NEU3 at 5 min and functions synergistically with TGF-β1. Human lung fibroblast cells were exposed to the indicated concentrations of ( A ) LAP alone and ( B ) suboptimal concentrations of LAP, TGF-β1, and a combination for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5–11 (4–6 male and 1–3 female cells lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control).

    Journal: Cells

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    doi: 10.3390/cells15050404

    Figure Lengend Snippet: Latency-associated peptide upregulates NEU3 at 5 min and functions synergistically with TGF-β1. Human lung fibroblast cells were exposed to the indicated concentrations of ( A ) LAP alone and ( B ) suboptimal concentrations of LAP, TGF-β1, and a combination for 5 min, fixed, and stained for NEU3 using immunofluorescence. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Values are normalized to 0 ng/mL control. Bars are mean ± SEM, n = 5–11 (4–6 male and 1–3 female cells lines). * p < 0.05, ** p < 0.01 (One-way ANOVA, Dunnet’s test compared to 0 ng/mL control).

    Article Snippet: Where indicated, recombinant human latent TGF-β1 (299-LT/CF, R&D systems), recombinant human latency associated peptide (LAP-H5213, Acro Biosystems, Newark, DE, USA), and lipopolysaccharide (437627, Caliobiochem Corporation, La Jolla, CA, USA) were diluted in protein free medium and added to cells.

    Techniques: Staining, Immunofluorescence, Control

    DDX3 mediates NEU3 upregulation by TGF-β1 and LAP. Human lung fibroblasts were pre-incubated with either 10 µM of RK-33 or an equal volume dilution of DMSO in protein free medium for 10 min. Protein free medium with or without ( A ) 10 ng/mL TGF-β1 or ( B ) 0.3 ng/mL LAP was added to wells for 5 min. Cells were then washed, fixed, and stained for NEU3 using immunofluorescence. RK-33 treated cells are indicated with red bars. Values are normalized to 0 min controls. Bars are mean ± SEM, n = 3–4 (1–2 male and 2 female cells lines). ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control). Human lung fibroblasts were treated with ( C – E ) 10 ng/mL of recombinant human active TGF-β1 or ( F – H ) 0.3 ng/mL of recombinant human LAP for the indicated times. m indicates minutes, h hour. The 0 min samples were treated with protein free media only. Phosphorylated proteins were isolated using TALON PMAC magnetic beads. Phosphorylated proteins and supernatants of total cell lysates were stained for DDX3 using Western blots. Blots are representative of five donors (four male and one female). ( D , E , G , H ) Quantification of Western blots. Band densities of phosphorylated and total DDX3 were normalized to the integrated density of silver- or Coomassie-stained gels respectively of total protein for each sample , then values were normalized to the 0 min control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM, n = 5 (4 male and 1 female). * p < 0.05, ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control).

    Journal: Cells

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    doi: 10.3390/cells15050404

    Figure Lengend Snippet: DDX3 mediates NEU3 upregulation by TGF-β1 and LAP. Human lung fibroblasts were pre-incubated with either 10 µM of RK-33 or an equal volume dilution of DMSO in protein free medium for 10 min. Protein free medium with or without ( A ) 10 ng/mL TGF-β1 or ( B ) 0.3 ng/mL LAP was added to wells for 5 min. Cells were then washed, fixed, and stained for NEU3 using immunofluorescence. RK-33 treated cells are indicated with red bars. Values are normalized to 0 min controls. Bars are mean ± SEM, n = 3–4 (1–2 male and 2 female cells lines). ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control). Human lung fibroblasts were treated with ( C – E ) 10 ng/mL of recombinant human active TGF-β1 or ( F – H ) 0.3 ng/mL of recombinant human LAP for the indicated times. m indicates minutes, h hour. The 0 min samples were treated with protein free media only. Phosphorylated proteins were isolated using TALON PMAC magnetic beads. Phosphorylated proteins and supernatants of total cell lysates were stained for DDX3 using Western blots. Blots are representative of five donors (four male and one female). ( D , E , G , H ) Quantification of Western blots. Band densities of phosphorylated and total DDX3 were normalized to the integrated density of silver- or Coomassie-stained gels respectively of total protein for each sample , then values were normalized to the 0 min control. Blue points indicate the averages for male cell lines and red points indicate the averages for female cells, circles represent healthy cell donors and triangles represent cells from IPF patients. Bars are mean ± SEM, n = 5 (4 male and 1 female). * p < 0.05, ** p < 0.01, *** p < 0.001 (One-way ANOVA, Dunnett’s test compared to 0 min control).

    Article Snippet: Where indicated, recombinant human latent TGF-β1 (299-LT/CF, R&D systems), recombinant human latency associated peptide (LAP-H5213, Acro Biosystems, Newark, DE, USA), and lipopolysaccharide (437627, Caliobiochem Corporation, La Jolla, CA, USA) were diluted in protein free medium and added to cells.

    Techniques: Incubation, Staining, Immunofluorescence, Control, Recombinant, Isolation, Magnetic Beads, Western Blot

    Proposed model of rapid NEU3 upregulation. Active TGF-β1 and LAP increase DDX3 interaction with NEU3 mRNA either by increasing binding to the common motif in NEU3 mRNA, found in 179 other proteins that are translationally regulated by TGF-β1 (orange), or to G-quadruplex structures (green; the image labels 4 of the 32 predicted G-quadruplex structures). DDX3 stimulates rapid NEU3 translation. Once translated and released from cells, NEU3 activates latent TGF-β1 (L-TGF-β1), releasing active TGF-β1 from its complex with LAP. TGF-β1 and LAP synergistically upregulate NEU3 translation leading to a positive feedback loop. DDX3 is inhibited by RK-33, and NEU3 is inhibited by 2-AP and AMPCA, all three of which can disrupt this loop and the rapid 5 min upregulation of NEU3. This figure was created using BioRender elements ( https://biorender.com/ (accessed on 16 July 2025)); structures of NEU3 and TGF-β1 were predicted using AlphaFold 3 .

    Journal: Cells

    Article Title: Latency-Associated Peptide Rapidly Upregulates Neuraminidase 3 in a Profibrotic Translation-Based Positive Feedback Loop

    doi: 10.3390/cells15050404

    Figure Lengend Snippet: Proposed model of rapid NEU3 upregulation. Active TGF-β1 and LAP increase DDX3 interaction with NEU3 mRNA either by increasing binding to the common motif in NEU3 mRNA, found in 179 other proteins that are translationally regulated by TGF-β1 (orange), or to G-quadruplex structures (green; the image labels 4 of the 32 predicted G-quadruplex structures). DDX3 stimulates rapid NEU3 translation. Once translated and released from cells, NEU3 activates latent TGF-β1 (L-TGF-β1), releasing active TGF-β1 from its complex with LAP. TGF-β1 and LAP synergistically upregulate NEU3 translation leading to a positive feedback loop. DDX3 is inhibited by RK-33, and NEU3 is inhibited by 2-AP and AMPCA, all three of which can disrupt this loop and the rapid 5 min upregulation of NEU3. This figure was created using BioRender elements ( https://biorender.com/ (accessed on 16 July 2025)); structures of NEU3 and TGF-β1 were predicted using AlphaFold 3 .

    Article Snippet: Where indicated, recombinant human latent TGF-β1 (299-LT/CF, R&D systems), recombinant human latency associated peptide (LAP-H5213, Acro Biosystems, Newark, DE, USA), and lipopolysaccharide (437627, Caliobiochem Corporation, La Jolla, CA, USA) were diluted in protein free medium and added to cells.

    Techniques: Binding Assay