mouse tm4sf5  (ProSci Incorporated)


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    ProSci Incorporated mouse tm4sf5
    Ab27 inhibits cancer cell growth by suppressing <t>TM4SF5-mediated</t> STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Mouse Tm4sf5, supplied by ProSci Incorporated, 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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    Average 93 stars, based on 1 article reviews
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    mouse tm4sf5 - by Bioz Stars, 2024-02
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    1) Product Images from "Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models"

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2022.01.006

    Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Figure Legend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Techniques Used: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

    Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).
    Figure Legend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Techniques Used: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

    Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.
    Figure Legend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Techniques Used: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

    Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in <xref ref-type=Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " title="... a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

    Techniques Used: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control

    mouse tm4sf5  (ProSci Incorporated)


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    ProSci Incorporated mouse tm4sf5
    Additional systemic overexpression of <t>TM4SF5</t> in Apc Min/+ mice led to intramucosal adenocarcinomas in the intestines. We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using hematoxylin and eosin (H&E) staining. Tissues from two representative animals are shown separately in combined images (A, B). We quantified the pathological conditions (C).
    Mouse Tm4sf5, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse tm4sf5 - by Bioz Stars, 2024-02
    86/100 stars

    Images

    1) Product Images from "Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis"

    Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2022.55.12.104

    Additional systemic overexpression of TM4SF5 in Apc Min/+ mice led to intramucosal adenocarcinomas in the intestines. We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using hematoxylin and eosin (H&E) staining. Tissues from two representative animals are shown separately in combined images (A, B). We quantified the pathological conditions (C).
    Figure Legend Snippet: Additional systemic overexpression of TM4SF5 in Apc Min/+ mice led to intramucosal adenocarcinomas in the intestines. We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using hematoxylin and eosin (H&E) staining. Tissues from two representative animals are shown separately in combined images (A, B). We quantified the pathological conditions (C).

    Techniques Used: Over Expression, Staining

    Additional systemic overexpression of TM4SF5 in Apc Min/+ mice increased β-catenin stabilization and transcriptional activity. (A) We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using immunohistochemistry (IHC) with mouse IgG or anti-β-catenin antibody. β-catenin immunostaining intensities were categorized as explained in the Materials and Methods section or quantitative comparison between conditions. (B) We analyzed HT29 cells via luciferase reporter assay following transfection with the β-catenin-responsive LEF/TCF-1 reporter pTOP-FLASH vector with shRNA plasmids (shControl or shTM4SF5) for 24 h; cells were treated with (+) or without (−) Wnt-3a for 12 h before the analysis. *, **, *** depict P < 0.05, 0.01, and 0.005, respectively. (C-E) HT29 (C, D) or HT116 (E) cells were independently transfected with control or Apc-full (C), shControl (shCon) or shTM4SF5 (D), or Mock-Flag or TM4SF5-Flag (E) plasmids for 24 h, and the cells were treated with recombinant Wnt-3a for 12 h, as explained in (B), prior to whole-cell extract preparation for standard Western blots for the indicated molecules. The data represent three independent experiments.
    Figure Legend Snippet: Additional systemic overexpression of TM4SF5 in Apc Min/+ mice increased β-catenin stabilization and transcriptional activity. (A) We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using immunohistochemistry (IHC) with mouse IgG or anti-β-catenin antibody. β-catenin immunostaining intensities were categorized as explained in the Materials and Methods section or quantitative comparison between conditions. (B) We analyzed HT29 cells via luciferase reporter assay following transfection with the β-catenin-responsive LEF/TCF-1 reporter pTOP-FLASH vector with shRNA plasmids (shControl or shTM4SF5) for 24 h; cells were treated with (+) or without (−) Wnt-3a for 12 h before the analysis. *, **, *** depict P < 0.05, 0.01, and 0.005, respectively. (C-E) HT29 (C, D) or HT116 (E) cells were independently transfected with control or Apc-full (C), shControl (shCon) or shTM4SF5 (D), or Mock-Flag or TM4SF5-Flag (E) plasmids for 24 h, and the cells were treated with recombinant Wnt-3a for 12 h, as explained in (B), prior to whole-cell extract preparation for standard Western blots for the indicated molecules. The data represent three independent experiments.

    Techniques Used: Over Expression, Activity Assay, Immunohistochemistry, Immunostaining, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, shRNA, Recombinant, Western Blot

    Apc Min/+ :Tg TM4SF5 mice showed sinusoidal dilatation, portal hypertension, and extracellular matrix (ECM) deposits in the liver. (A-C) We analyzed liver tissues from wild-type (WT, n = 4), Apc Min/+ ( n = 4), or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using IHC with normal IgG, anti-TM4SF5, or anti-β-catenin antibody (A) and H&E staining or Masson’s trichrome staining (B). The liver tissues were also immunoblotted for the indicated molecules (C). (D) TM4SF5-null SNU449 or endogenously TM4SF5-expressing Hep3B hepatocytes were transfected with control, TM4SF5 WT, shControl, or shTM4SF5 #4 plasmids for 48 h, before whole cell lysate preparation for immunoblots for the indicated molecules. The data represent three independent experiments.
    Figure Legend Snippet: Apc Min/+ :Tg TM4SF5 mice showed sinusoidal dilatation, portal hypertension, and extracellular matrix (ECM) deposits in the liver. (A-C) We analyzed liver tissues from wild-type (WT, n = 4), Apc Min/+ ( n = 4), or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using IHC with normal IgG, anti-TM4SF5, or anti-β-catenin antibody (A) and H&E staining or Masson’s trichrome staining (B). The liver tissues were also immunoblotted for the indicated molecules (C). (D) TM4SF5-null SNU449 or endogenously TM4SF5-expressing Hep3B hepatocytes were transfected with control, TM4SF5 WT, shControl, or shTM4SF5 #4 plasmids for 48 h, before whole cell lysate preparation for immunoblots for the indicated molecules. The data represent three independent experiments.

    Techniques Used: Staining, Expressing, Transfection, Western Blot

    Tg TM4SF5 mice at 1.5 years old showed the phenotypes of portal hypertension associated with steatohepatitis and ECM deposits. We collected and analyzed liver tissues from 1.5-year-old C57BL/6 WT or Tg TM4SF5 male mice (n = 7) using H&E staining and Masson’s trichrome staining. Random representative images are shown. Image magnification is shown at 100× or 400×.
    Figure Legend Snippet: Tg TM4SF5 mice at 1.5 years old showed the phenotypes of portal hypertension associated with steatohepatitis and ECM deposits. We collected and analyzed liver tissues from 1.5-year-old C57BL/6 WT or Tg TM4SF5 male mice (n = 7) using H&E staining and Masson’s trichrome staining. Random representative images are shown. Image magnification is shown at 100× or 400×.

    Techniques Used: Staining

    mouse tm4sf5  (ProSci Incorporated)


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    ProSci Incorporated mouse tm4sf5
    Ab27 inhibits cancer cell growth by suppressing <t>TM4SF5-mediated</t> STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Mouse Tm4sf5, supplied by ProSci Incorporated, 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/result/mouse tm4sf5/product/ProSci Incorporated
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse tm4sf5 - by Bioz Stars, 2024-02
    93/100 stars

    Images

    1) Product Images from "Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models"

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2022.01.006

    Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Figure Legend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Techniques Used: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

    Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).
    Figure Legend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Techniques Used: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

    Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.
    Figure Legend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Techniques Used: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

    Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in <xref ref-type=Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " title="... a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

    Techniques Used: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control

    rabbit anti mouse tm4sf5  (ProSci Incorporated)


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    Structured Review

    ProSci Incorporated rabbit anti mouse tm4sf5
    Ab27 inhibits cancer cell growth by suppressing <t>TM4SF5-mediated</t> STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Rabbit Anti Mouse Tm4sf5, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mouse tm4sf5/product/ProSci Incorporated
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mouse tm4sf5 - by Bioz Stars, 2024-02
    86/100 stars

    Images

    1) Product Images from "Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models"

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2022.01.006

    Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Figure Legend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Techniques Used: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

    Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).
    Figure Legend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Techniques Used: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

    Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.
    Figure Legend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Techniques Used: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

    Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in <xref ref-type=Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " title="... a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

    Techniques Used: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control

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    ProSci Incorporated mouse tm4sf5
    Ab27 inhibits cancer cell growth by suppressing <t>TM4SF5-mediated</t> STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
    Mouse Tm4sf5, supplied by ProSci Incorporated, 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/result/mouse tm4sf5/product/ProSci Incorporated
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse tm4sf5 - by Bioz Stars, 2024-02
    93/100 stars
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    86
    ProSci Incorporated rabbit anti mouse tm4sf5
    Ab27 inhibits cancer cell growth by suppressing <t>TM4SF5-mediated</t> STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.
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    Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

    Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

    Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

    Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in <xref ref-type=Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " width="100%" height="100%">

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control

    Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Staining, Binding Assay, Activity Assay, Incubation, Growth Assay

    Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Staining

    Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection

    Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in <xref ref-type=Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F). " width="100%" height="100%">

    Journal: Molecular Therapy Oncolytics

    Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

    doi: 10.1016/j.omto.2022.01.006

    Figure Lengend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

    Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

    Techniques: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Staining, Binding Assay, Injection, Negative Control