Structured Review

GeneTex cd81
Interaction of GPC3 with <t>CD81.</t> A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
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Images

1) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

2) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

3) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

4) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

5) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

6) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

7) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

8) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

9) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

10) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

11) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

12) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

13) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

14) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

15) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

16) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

17) Product Images from "Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte"

Article Title: Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-019-1177-1

Authentication of ADSCs-derived exosome. a Purity identification of ADSCs-Exo by flow cytometry using CD9, CD63, CD81, and their isotype control antibodies. b Measurement of protein level of CD9, CD63, and CD81 by western blotting. c Images of exosome morphology taking by transmission electron microscopy (TEM). d Observation of number and size of exosome by nanoparticle tracking analysis. ADSCs-Exo indicates ADSCs-derived exosome
Figure Legend Snippet: Authentication of ADSCs-derived exosome. a Purity identification of ADSCs-Exo by flow cytometry using CD9, CD63, CD81, and their isotype control antibodies. b Measurement of protein level of CD9, CD63, and CD81 by western blotting. c Images of exosome morphology taking by transmission electron microscopy (TEM). d Observation of number and size of exosome by nanoparticle tracking analysis. ADSCs-Exo indicates ADSCs-derived exosome

Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Western Blot, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

18) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

19) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

20) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

21) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

22) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

23) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

24) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

25) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

26) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

27) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

28) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

29) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

30) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

31) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

32) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

33) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

34) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

35) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

36) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

37) Product Images from "Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation"

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.081129

Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining
Figure Legend Snippet: Immunofluorescence analysis of GPC3 and CD81 expression in rat liver regeneration process. Olympus Fluoview 500 confocal microscope was used to visualize the images; original magnification, × 200. GPC3 (green), CD81 (red), and DRAQ5 nuclease staining

Techniques Used: Immunofluorescence, Expressing, Microscopy, Staining

Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
Figure Legend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

Techniques Used: Immunoprecipitation, Incubation, Western Blot

RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81
Figure Legend Snippet: RNA and protein levels of GPC3 and CD81 increase during liver regeneration. A: Semiquantitative RT-PCR results show that GPC3 and CD81 expression are up-regulated in PHx after day 2. GAPDH: loading control. NRT: non-RT negative control. B: GPC3 and CD81

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

38) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

39) Product Images from "Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex"

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.03.013

Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
Figure Legend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay

Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.
Figure Legend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Negative Control

Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.
Figure Legend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

Techniques Used: Binding Assay, Activation Assay, Transmission Electron Microscopy

Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P
Figure Legend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

Techniques Used: Western Blot, Immunoprecipitation, Mouse Assay, Staining

40) Product Images from "Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes"

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.02.013

Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.
Figure Legend Snippet: Hepatitis C virus (HCV) E2 protein enhances Hippo activity and decreases yes-associated protein (Yap) in human hepatocellular carcinoma and primary human hepatocytes. A: HepaRG cells were treated with HCV E2 protein 10 μg/mL for 5 hours, and whole-cell lysates were analyzed by Western blot for CD81, MPS one binder kinase activator-like protein (Mob)-1, phosphorylated (p)-MOB1, serine/threonine-protein kinase LATS-1, p-LATS1, and YAP/tafazzin (Taz). B: Primary human hepatocytes were treated with 10 μg/mL HCV E2 protein for 5 hours, and total nuclear protein and cytoplasmic fraction were analyzed separately by Western blot for YAP, CD81, ezrin, and p-ezrin (Thr567) and cleaved (activated) p–mammalian STE20-like protein kinase (MST)-1.

Techniques Used: Activity Assay, Western Blot, Microscale Thermophoresis

Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.
Figure Legend Snippet: Forced expression of CD81 in glypican (Gpc)-3–expressing JM2 hepatoma cells results in activation of the Hippo pathway, a decrease in nuclear Yap, and a decrease in the percentage of bromodeoxyuridine (BrdU)-positive nuclei. JM2 cells were transiently transfected with pCMV6-CD81 plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis. A: Expression of CD81, Gpc3, ezrin, and phosphorylated (p)-ezrin (Thr567). B: Cleaved (activated) p-mammalian STE20-like protein kinase (Mst)-1, yes-associated protein (Yap), and p-Yap. In both A and B , each band represents a separate cell culture. Results are representative of four experiments. C: JM2 cells in sparse density (50%) after 24 hours of transfection and 24 hours of BrdU incorporation. Positive ratio indicates the fraction of cells whose nuclei were positive for BrdU by IHC analysis. Data are expressed as means ± SEM, derived from four separate cultures for each category ( C ). P = 0.0078.

Techniques Used: Expressing, Gel Permeation Chromatography, Activation Assay, Transfection, Plasmid Preparation, Microscale Thermophoresis, Cell Culture, BrdU Incorporation Assay, Immunohistochemistry, Derivative Assay

Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .
Figure Legend Snippet: Inhibition of CD81-associated tyrosine-protein kinase SYK (Syk) results in decrease in yes-associated protein (Yap) expression and ezrin phosphorylation. A: JM2 rat hepatocellular carcinoma cells were treated with Syk inhibitor at 0.1 and 0.4 μg/mL for 5 hours, and total proteins were analyzed by Western blot for Yap, ezrin, and phosphorylated (p)-ezrin (Thr567). B: JM2 cells were transiently transfected with pCMV6-Syk plasmid, and after 12 hours, total cell lysates were collected for protein expression analysis of Syk, ezrin, and p-ezrin (Thr567), cleaved p–mammalian STE20-like protein kinase (Mst)-1, Yap/tafazzin (Taz), and p-Yap. Red indicates high saturation of the band. C: Quantitative analysis of relative percentage of p-Yap and Yap expression from C .

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Microscale Thermophoresis

Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P
Figure Legend Snippet: Effects of the CD81 agonistic antibody 5A6 on hepatocyte proliferation. Rat hepatocytes at day 6 in primary culture were stimulated with 1 μg/mL of 5A6 or inactivated control for 5 hours. Yes-associated protein (Yap) and tafazzin (Taz) expression levels in nuclear extracts ( left panels ), total ezrin and phosphorylated (p)-ezrin (Thr567) expression levels in cytoplasm ( right panels ) were detected by Western blot analysis. Each bar indicates the ratio between the intensity of the relative band divided by the intensity of the Ponceau stain. The bars in the graphs in the bottom panels correspond to the labels and conditions shown in the corresponding top panels for the Western gels ( white bars , control medium; black bars , inactivated 5A6 agonistic antibody; red bars , 5A6 agonistic antibody; blue bars , 5A6 agonistic antibody+HGF). Data are expressed as means ± SEM of four separate cultures. ∗ P

Techniques Used: Expressing, Western Blot, Staining

Related Articles

Activation Assay:

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes
Article Snippet: .. The overall results demonstrate that the HCV E2 protein interacting with the CD81 mimics the effects of GPC3 and results in decreased p-ezrin, enhanced activation of the MST1 and LATS1 kinases of the Hippo pathway, and decrease in nuclear YAP. ..

Incubation:

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex
Article Snippet: .. Lysates incubated with GST protein were unable to pull-down CD81 ( , C and D), confirming that CD81 is a specific binding partner for Hhex and not GST. .. Thus, using two completely different techniques and using both proteins as baits, we were able to show that CD81 and Hhex are binding partners for each other.

other:

Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation
Article Snippet: Several interesting genes were found, such as tyrosine kinase substrate (Hrs), prostaglandin D2 synthase (Ptgds), α-2-HS-glycoprotein (fetuin), vitamin D-binding protein, splicing factor (SRp20), ribosomal protein S2 (Rps2), ribosomal protein L29, T-cell differentiation protein 2(Mal2), 5,10-methenyltetrahydrofolate synthetase (Mthfs), α-1-inhibitor III, argininosuccinate synthetase (Ass), afamin (Afm), and CD81.

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes
Article Snippet: Treatment with HCV E2 protein did not have any effect on the levels of CD81 itself or total ezrin (data not shown), in either HepaRG or primary human hepatocytes.

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex
Article Snippet: Confirming previous findings from our laboratory in the rat, we found that GPC3 binds to CD81 by co-immunoprecipitation in mouse as well ( A).

Article Title: Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes
Article Snippet: At present, there is no mechanistic correlation between CD81 and direct regulation of GPC3.

Binding Assay:

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex
Article Snippet: .. Lysates incubated with GST protein were unable to pull-down CD81 ( , C and D), confirming that CD81 is a specific binding partner for Hhex and not GST. .. Thus, using two completely different techniques and using both proteins as baits, we were able to show that CD81 and Hhex are binding partners for each other.

Mouse Assay:

Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex
Article Snippet: .. There was significantly more CD81 bound to GPC3 in TG mice compared with WT mice at all time points ( , A and B). .. Within the group (WT and TG mice), the amount of CD81 bound to GPC3 significantly decreased on day 2 (proliferation peak) and increased by day 8 (past end of hepatocyte proliferation).

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    GeneTex cd81
    Interaction of GPC3 with <t>CD81.</t> A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P
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    Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

    Journal: The American Journal of Pathology

    Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

    doi: 10.1016/j.ajpath.2013.03.013

    Figure Lengend Snippet: Interaction of GPC3 with CD81. A : Western blot of CD81 and GPC3 after co-immunoprecipitation using anti-GPC3 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound CD81 normalized to the amount of GPC3 pulled down over a time course after PHx in WT and TG mice. ∗ P

    Article Snippet: Primary antibodies used with their concentrations were as follows: GPC3 ( Aviva Systems Biology Corp., San Diego, CA, 1:1000; SC10456, Santa Cruz Biotechnology, Dallas, TX, 1:200), CD81 (GTX75432 GeneTex, Irvine, CA, 1:1000; SC70803, Santa Cruz, 1:500), Indian HH (IHH) (ab52919, Abcam, Cambridge, MA, 1:5000), Smoothened (ab72130, Abcam, 1:2000), GLI1 ( , Abnova, Taipei City, Taiwan, 1:2000), patched-1 (ab39266, 1:2000; sc6147, 1:250), and Hhex (CA1326, Cell Applications, San Diego, CA).

    Techniques: Western Blot, Immunoprecipitation, Mouse Assay

    Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

    Journal: The American Journal of Pathology

    Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

    doi: 10.1016/j.ajpath.2013.03.013

    Figure Lengend Snippet: Hhex-GST pull-down assay. A : Western blot analysis of pull-down of Hhex-GST fusion protein confirmed using anti-Hhex antibody. B : CD81 detected on the same membrane using anti-CD81 antibody, indicating it to be a binding partner for Hhex. C : GST protein detected after using GST protein instead of HHEX-GST fusion protein for the assay as a negative control. D : CD81 could not be detected on the same membrane, confirming the specificity of CD81 binding to Hhex.

    Article Snippet: Primary antibodies used with their concentrations were as follows: GPC3 ( Aviva Systems Biology Corp., San Diego, CA, 1:1000; SC10456, Santa Cruz Biotechnology, Dallas, TX, 1:200), CD81 (GTX75432 GeneTex, Irvine, CA, 1:1000; SC70803, Santa Cruz, 1:500), Indian HH (IHH) (ab52919, Abcam, Cambridge, MA, 1:5000), Smoothened (ab72130, Abcam, 1:2000), GLI1 ( , Abnova, Taipei City, Taiwan, 1:2000), patched-1 (ab39266, 1:2000; sc6147, 1:250), and Hhex (CA1326, Cell Applications, San Diego, CA).

    Techniques: Pull Down Assay, Western Blot, Binding Assay, Negative Control

    Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

    Journal: The American Journal of Pathology

    Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

    doi: 10.1016/j.ajpath.2013.03.013

    Figure Lengend Snippet: Hypothesized mechanism(s) for GPC3 as a growth regulator. In resting liver, GPC3 regulates growth by two pathways: it competes with patched for HH binding, thus inhibiting nuclear activation of GLI, and it binds to CD81, thus decreasing the availability of free CD81 to bind to Hhex and keeping it outside the nucleus. In regenerating liver, GPC3 binding to HH and CD81 decreases, which leads to induction of HH signaling and increased CD81-Hhex binding. Hence, GPC3 brings about increased nuclear GLI and decreased nuclear Hhex during cell proliferation. SMO, smoothened; TEM, tetraspanin enhanced microdomain.

    Article Snippet: Primary antibodies used with their concentrations were as follows: GPC3 ( Aviva Systems Biology Corp., San Diego, CA, 1:1000; SC10456, Santa Cruz Biotechnology, Dallas, TX, 1:200), CD81 (GTX75432 GeneTex, Irvine, CA, 1:1000; SC70803, Santa Cruz, 1:500), Indian HH (IHH) (ab52919, Abcam, Cambridge, MA, 1:5000), Smoothened (ab72130, Abcam, 1:2000), GLI1 ( , Abnova, Taipei City, Taiwan, 1:2000), patched-1 (ab39266, 1:2000; sc6147, 1:250), and Hhex (CA1326, Cell Applications, San Diego, CA).

    Techniques: Binding Assay, Activation Assay, Transmission Electron Microscopy

    Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

    Journal: The American Journal of Pathology

    Article Title: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

    doi: 10.1016/j.ajpath.2013.03.013

    Figure Lengend Snippet: Interaction of CD81 with Hhex. A : Western blot of Hhex and CD81 after co-immunoprecipitation using anti-CD81 antibody in WT and GPC3 TG mice at days 0, 2, and 8 after PHx. B : Ratios of densitometry of bound Hhex normalized to the amount of CD81 pulled down during a time after PHx in WT and TG mice. C : Western blot of nuclear Hhex and Ponceau stain as loading control in WT and GPC3 TG mice during time after PHx. D : Densitometric analyses of nuclear Hhex normalized to Ponceau stain. ∗ P

    Article Snippet: Primary antibodies used with their concentrations were as follows: GPC3 ( Aviva Systems Biology Corp., San Diego, CA, 1:1000; SC10456, Santa Cruz Biotechnology, Dallas, TX, 1:200), CD81 (GTX75432 GeneTex, Irvine, CA, 1:1000; SC70803, Santa Cruz, 1:500), Indian HH (IHH) (ab52919, Abcam, Cambridge, MA, 1:5000), Smoothened (ab72130, Abcam, 1:2000), GLI1 ( , Abnova, Taipei City, Taiwan, 1:2000), patched-1 (ab39266, 1:2000; sc6147, 1:250), and Hhex (CA1326, Cell Applications, San Diego, CA).

    Techniques: Western Blot, Immunoprecipitation, Mouse Assay, Staining