antibodies cd81  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc antibodies cd81
    The sEVs from CAFs increased breast cancer cell survival and metastasis ability. A The representative picture of sEVs from NFs or CAFs. Transmission electron microscopy was used to take the pictures. B The sEVs markers were identified. The total protein was isolated from NFs or CAFs derived sEVs and performed for <t>CD81,</t> CD63, and GM130 immunoblotting by western blotting. C , D Cell proliferation of MDA-MB-231 and SKBR3 cells exposed to CAFs-sEV and NFs-sEV (25 µg/ml) assayed by CCK8. E Wound healing assay was used to examine cell migration of MDA-MB-231 and SKBR3 cells with CAFs-sEV or NFs-sEV treatment (25ug/ml) (scale bars, 100 µm). F , G Data analysis of ( E ). H Transwell system was used to examine cell invasion of MDA-MB-231 and SKBR3 cells with CAFs-sEV or NFs-sEV treatment (25 µg/ml) (scale bars, 100 µm). I , J Data analysis of ( H ). K Tumor growth in vivo. l the average tumor weight. M IHC straining for Ki67 (scale bars, 50 µm). * p
    Antibodies Cd81, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Carcinoma associated fibroblasts small extracellular vesicles with low miR-7641 promotes breast cancer stemness and glycolysis by HIF-1α"

    Article Title: Carcinoma associated fibroblasts small extracellular vesicles with low miR-7641 promotes breast cancer stemness and glycolysis by HIF-1α

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-021-00524-x

    The sEVs from CAFs increased breast cancer cell survival and metastasis ability. A The representative picture of sEVs from NFs or CAFs. Transmission electron microscopy was used to take the pictures. B The sEVs markers were identified. The total protein was isolated from NFs or CAFs derived sEVs and performed for CD81, CD63, and GM130 immunoblotting by western blotting. C , D Cell proliferation of MDA-MB-231 and SKBR3 cells exposed to CAFs-sEV and NFs-sEV (25 µg/ml) assayed by CCK8. E Wound healing assay was used to examine cell migration of MDA-MB-231 and SKBR3 cells with CAFs-sEV or NFs-sEV treatment (25ug/ml) (scale bars, 100 µm). F , G Data analysis of ( E ). H Transwell system was used to examine cell invasion of MDA-MB-231 and SKBR3 cells with CAFs-sEV or NFs-sEV treatment (25 µg/ml) (scale bars, 100 µm). I , J Data analysis of ( H ). K Tumor growth in vivo. l the average tumor weight. M IHC straining for Ki67 (scale bars, 50 µm). * p
    Figure Legend Snippet: The sEVs from CAFs increased breast cancer cell survival and metastasis ability. A The representative picture of sEVs from NFs or CAFs. Transmission electron microscopy was used to take the pictures. B The sEVs markers were identified. The total protein was isolated from NFs or CAFs derived sEVs and performed for CD81, CD63, and GM130 immunoblotting by western blotting. C , D Cell proliferation of MDA-MB-231 and SKBR3 cells exposed to CAFs-sEV and NFs-sEV (25 µg/ml) assayed by CCK8. E Wound healing assay was used to examine cell migration of MDA-MB-231 and SKBR3 cells with CAFs-sEV or NFs-sEV treatment (25ug/ml) (scale bars, 100 µm). F , G Data analysis of ( E ). H Transwell system was used to examine cell invasion of MDA-MB-231 and SKBR3 cells with CAFs-sEV or NFs-sEV treatment (25 µg/ml) (scale bars, 100 µm). I , J Data analysis of ( H ). K Tumor growth in vivo. l the average tumor weight. M IHC straining for Ki67 (scale bars, 50 µm). * p

    Techniques Used: Transmission Assay, Electron Microscopy, Isolation, Derivative Assay, Western Blot, Multiple Displacement Amplification, Wound Healing Assay, Migration, In Vivo, Immunohistochemistry

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    Cell Signaling Technology Inc antibody cd8
    Fluorescent multiplex immunohistochemistry (mIHC) staining pattern for tumour cell PD-L1 and TILs in gastric adenocarcinoma tissues ( A ) Strong expression of PD-L1 on tumour cells with high density of tumour infiltrating CD3+ and <t>CD8+</t> cells (original magnification × 100). ( B ) Strong expression of PD-L1 on tumour cells with high density of tumour infiltrating CD3+ cells (white arrow) and CD3+CD8+ cells (white arrowhead) (original magnification × 400). ( C ) Negative expression PD-L1 on tumour cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( D ) Negative expression PD-L1 on tumour cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 400).
    Antibody Cd8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cd8
    Effects of CTP on CD4, <t>CD8,</t> IFN-γ, and IL-4 of the spleen and colorectal tumor in Apc Min/+ mice. CTP enhanced the levels of CD4, CD8, and IFN-γ, and suppressed the levels of IL-4 in (A) the spleen (400 ×, scale bar: 20 μm) and (B) colorectal tumor (200 ×, scale bar: 50 μm) analyzed by immunohistochemistry. The pixel density for the semi-quantitative densitometric analysis of protein expressions in (C) the spleen and (D) colorectal tumors were quantified. Data are presented as mean ± S.D. and analyzed via a one-way ANOVA test ( n = 3). # p
    Cd8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc immunoblot analysis
    Validation of shRNA screen hits in H1650 and H1650-M3 cells using the clonogenic assay technique. ( A ) Clonogenic assay performed on H1650 and H1650-M3 to validate siRNA screen hits for BRCA1 , ORC5L , RFC3 , and POLS . The charts represent the percentage of viable cells 15 days after transfection with the indicated siRNA oligonucleotides compared to that of scramble-siRNA-transfected control cells. Each bar represents mean ± SD of data collected from ten fields from three different six-well plates (n = 30). ( B ) Knockdown efficiency for clonogenic assay upon transfection with the indicated siRNA oligonucleotides. Samples were collected 3 days post-transfection and analyzed by <t>immunoblot</t> analysis with antibodies against Total caspase 3, alpha-tubulin and Cleaved-caspase 3. Each bar represents mean ± SD of three replicates. p-value *
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    Image Search Results


    Fluorescent multiplex immunohistochemistry (mIHC) staining pattern for tumour cell PD-L1 and TILs in gastric adenocarcinoma tissues ( A ) Strong expression of PD-L1 on tumour cells with high density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( B ) Strong expression of PD-L1 on tumour cells with high density of tumour infiltrating CD3+ cells (white arrow) and CD3+CD8+ cells (white arrowhead) (original magnification × 400). ( C ) Negative expression PD-L1 on tumour cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( D ) Negative expression PD-L1 on tumour cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 400).

    Journal: Oncotarget

    Article Title: Predictive relevance of PD-L1 expression with pre-existing TILs in gastric cancer

    doi: 10.18632/oncotarget.22079

    Figure Lengend Snippet: Fluorescent multiplex immunohistochemistry (mIHC) staining pattern for tumour cell PD-L1 and TILs in gastric adenocarcinoma tissues ( A ) Strong expression of PD-L1 on tumour cells with high density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( B ) Strong expression of PD-L1 on tumour cells with high density of tumour infiltrating CD3+ cells (white arrow) and CD3+CD8+ cells (white arrowhead) (original magnification × 400). ( C ) Negative expression PD-L1 on tumour cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( D ) Negative expression PD-L1 on tumour cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 400).

    Article Snippet: For Fluorescent Multiplex immunohistochemistry (mIHC), firstly, slides were incubated in 1:250 diluted primary antibody CD8 (clone: C8/144B, PD-L1/CD3ε/ CD8α /Multiplex IHC Antibody Panel #65713, Cell Signaling Technology, MA USA.) for 60 minutes at room temperature.

    Techniques: Fluorescent Multiplex Immunohistochemistry, Staining, Expressing

    Kaplan-Meier survival analysis with Log-Rank test of PD-L1 combined with TILs ( A ) Survival curves of positive and negative expression of PD-L1 on tumour cells with low density and high density tumour infiltrating CD3+ cells. ( B ) Survival curves of positive and negative expression of PD-L1 on tumour cells with low density and high density tumour infiltrating CD8+ cells. ( C ) Survival curves of positive and negative expression of PD-L1 on immune cells with low density and high density tumour infiltrating CD3+ cells. ( D ) Survival curves of positive and negative expression of PD-L1 on immune cells with low density and high density tumour infiltrating CD8+cells.

    Journal: Oncotarget

    Article Title: Predictive relevance of PD-L1 expression with pre-existing TILs in gastric cancer

    doi: 10.18632/oncotarget.22079

    Figure Lengend Snippet: Kaplan-Meier survival analysis with Log-Rank test of PD-L1 combined with TILs ( A ) Survival curves of positive and negative expression of PD-L1 on tumour cells with low density and high density tumour infiltrating CD3+ cells. ( B ) Survival curves of positive and negative expression of PD-L1 on tumour cells with low density and high density tumour infiltrating CD8+ cells. ( C ) Survival curves of positive and negative expression of PD-L1 on immune cells with low density and high density tumour infiltrating CD3+ cells. ( D ) Survival curves of positive and negative expression of PD-L1 on immune cells with low density and high density tumour infiltrating CD8+cells.

    Article Snippet: For Fluorescent Multiplex immunohistochemistry (mIHC), firstly, slides were incubated in 1:250 diluted primary antibody CD8 (clone: C8/144B, PD-L1/CD3ε/ CD8α /Multiplex IHC Antibody Panel #65713, Cell Signaling Technology, MA USA.) for 60 minutes at room temperature.

    Techniques: Expressing

    Fluorescent multiplex immunohistochemistry (mIHC) staining pattern for immune cell PD-L1 and TILs in gastric adenocarcinoma tissues ( A ) Negative expression of PD-L1 on immnune cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( B ) Positive expression of PD-L1 on immune cells with high density of tumour infiltrating CD3+ cells and CD8+ cells (original magnification ×100). ( C ) Positive expression PD-L1 on tumour cells with low density of tumour infiltrating PD-L1+CD3+ (white arrow) and PD-L1+CD3+CD8+ cells (white arrowhead) (original magnification × 400).

    Journal: Oncotarget

    Article Title: Predictive relevance of PD-L1 expression with pre-existing TILs in gastric cancer

    doi: 10.18632/oncotarget.22079

    Figure Lengend Snippet: Fluorescent multiplex immunohistochemistry (mIHC) staining pattern for immune cell PD-L1 and TILs in gastric adenocarcinoma tissues ( A ) Negative expression of PD-L1 on immnune cells with low density of tumour infiltrating CD3+ and CD8+ cells (original magnification × 100). ( B ) Positive expression of PD-L1 on immune cells with high density of tumour infiltrating CD3+ cells and CD8+ cells (original magnification ×100). ( C ) Positive expression PD-L1 on tumour cells with low density of tumour infiltrating PD-L1+CD3+ (white arrow) and PD-L1+CD3+CD8+ cells (white arrowhead) (original magnification × 400).

    Article Snippet: For Fluorescent Multiplex immunohistochemistry (mIHC), firstly, slides were incubated in 1:250 diluted primary antibody CD8 (clone: C8/144B, PD-L1/CD3ε/ CD8α /Multiplex IHC Antibody Panel #65713, Cell Signaling Technology, MA USA.) for 60 minutes at room temperature.

    Techniques: Fluorescent Multiplex Immunohistochemistry, Staining, Expressing

    Kaplan-Meier survival analysis with Log-Rank test PD-L1 and TILs ( A ) Survival curves of PD-L1 positive and negative in tumour cells. ( B ) Survival curves of PD-L1 positive and negative in immune cells. ( C ) Survival curves of low-density and high-density CD3+ TILs. ( D ) Survival curves of low-density and high-density CD8+ TILs.

    Journal: Oncotarget

    Article Title: Predictive relevance of PD-L1 expression with pre-existing TILs in gastric cancer

    doi: 10.18632/oncotarget.22079

    Figure Lengend Snippet: Kaplan-Meier survival analysis with Log-Rank test PD-L1 and TILs ( A ) Survival curves of PD-L1 positive and negative in tumour cells. ( B ) Survival curves of PD-L1 positive and negative in immune cells. ( C ) Survival curves of low-density and high-density CD3+ TILs. ( D ) Survival curves of low-density and high-density CD8+ TILs.

    Article Snippet: For Fluorescent Multiplex immunohistochemistry (mIHC), firstly, slides were incubated in 1:250 diluted primary antibody CD8 (clone: C8/144B, PD-L1/CD3ε/ CD8α /Multiplex IHC Antibody Panel #65713, Cell Signaling Technology, MA USA.) for 60 minutes at room temperature.

    Techniques:

    Five specific immune infiltrating cells contribute to build the prognostic model. (A) Five immune infiltrating cells including Neut (red), Masta (purple), macrophages (black), NKa (light blue), CD8T (dark blue) were selected by LASSO Cox regression method. (B) IS, survival status and (C) OS time of patients in Cohort 1. (D) Heatmap of 5 immune infiltrating cells profiles (the transition from pink to blue suggested the transition from low proportion to high proportion). (E) Scatter plots showed cluster 2 had lower IS than cluster 1 and 3. (F) The histogram of immune type in 3 clusters. The histogram of (G) FIGO staging, (H) metastasis in IS Lo and IS Hi. (I) Violin plots of immune infiltrating cells between the 2 groups. CD8T, CD8 T cells; FIGO, International Federation of Gynecology and Obstetrics; IS, immunoscore; LASSO, least absolute shrinkage and selection operator; Masta, mast activated cells; M0, M0 macrophages; Neut, neutrophils; NKa, NK activated cells; OS, overall survival. ns, not significant; * p

    Journal: Journal of Gynecologic Oncology

    Article Title: The prognostic significance of tumor-infiltrating lymphocytes in cervical cancer

    doi: 10.3802/jgo.2021.32.e32

    Figure Lengend Snippet: Five specific immune infiltrating cells contribute to build the prognostic model. (A) Five immune infiltrating cells including Neut (red), Masta (purple), macrophages (black), NKa (light blue), CD8T (dark blue) were selected by LASSO Cox regression method. (B) IS, survival status and (C) OS time of patients in Cohort 1. (D) Heatmap of 5 immune infiltrating cells profiles (the transition from pink to blue suggested the transition from low proportion to high proportion). (E) Scatter plots showed cluster 2 had lower IS than cluster 1 and 3. (F) The histogram of immune type in 3 clusters. The histogram of (G) FIGO staging, (H) metastasis in IS Lo and IS Hi. (I) Violin plots of immune infiltrating cells between the 2 groups. CD8T, CD8 T cells; FIGO, International Federation of Gynecology and Obstetrics; IS, immunoscore; LASSO, least absolute shrinkage and selection operator; Masta, mast activated cells; M0, M0 macrophages; Neut, neutrophils; NKa, NK activated cells; OS, overall survival. ns, not significant; * p

    Article Snippet: IHC and evaluationFor IHC, tissue microarray (TMA) sections were performed through deparaffinizing, rehydrating, bringing slides to a boiled sodium citrate buffer then cooling down to room temperature, blocking each section with 100–200 µL blocking solution for 2 hour at room temperature, incubating primary antibodies with PD-1 (HPA035981, 1:100; Sigma, St. Louis, MO, USA), PD-L1 (SAB4301882, 1:60; Sigma), GZMB (46890s, 1:500; Cell Signaling Technology, Danvers, MA, USA), CD68 (ab955, 1:300; Abcam, Cambridge, UK), CD56 (07-5603, 1:600; Invitrogen, Carlsbad, CA, USA), CD8a (70306s, 1:200; Cell Signaling Technology), anti-Mast cell tryptase (ab2378, 1:10,000; Abcam), CD66b (392902, 1:400; Biolegend, San Diego, CA, USA) overnight at 4°C, adding secondary antibody and staining.

    Techniques: Selection

    Clinical information and survival analysis of 3 clusters based on immune infiltrates. (A) Tumor infiltrating cells, clinical and pathological features of 138 samples for 3 clusters in Cohort 1. (B) Kaplan-Meier survival curves displayed the clinical outcomes of 3 clusters. ACC, adenocarcinoma; Bm, B memory cells; Bn, B naïve cells; CD4Tma, activated memory CD4 T cells; CD4Tmr, resting memory CD4 T cellls; CD4Tn, naïve CD4 T cells; CD8T, CD8 T cells; DCa, activated dendritic cells; DCr, resting dendritic cells; Eos, eosinophils; Fibro, fibrocytes; FIGO, International Federation of Gynecology and Obstetrics; HPV, human papillomavirus; M, metastasis; Masta, mast activated cells; Mastr, resting mast cells; Mono, monocytes; M0, M0 macrophages; M1, M1 macrophages; M2, M2 macrophages; N, node; Neut, neutrophils; NKa, NK activated cells; NKr, resting NK cells; Plasma, plasma cells; SCC, squamous cell carcinoma; T, tumor; Tfh, follicular helper T cells; Tgd, γδT cells; Treg, regulatory T cells.

    Journal: Journal of Gynecologic Oncology

    Article Title: The prognostic significance of tumor-infiltrating lymphocytes in cervical cancer

    doi: 10.3802/jgo.2021.32.e32

    Figure Lengend Snippet: Clinical information and survival analysis of 3 clusters based on immune infiltrates. (A) Tumor infiltrating cells, clinical and pathological features of 138 samples for 3 clusters in Cohort 1. (B) Kaplan-Meier survival curves displayed the clinical outcomes of 3 clusters. ACC, adenocarcinoma; Bm, B memory cells; Bn, B naïve cells; CD4Tma, activated memory CD4 T cells; CD4Tmr, resting memory CD4 T cellls; CD4Tn, naïve CD4 T cells; CD8T, CD8 T cells; DCa, activated dendritic cells; DCr, resting dendritic cells; Eos, eosinophils; Fibro, fibrocytes; FIGO, International Federation of Gynecology and Obstetrics; HPV, human papillomavirus; M, metastasis; Masta, mast activated cells; Mastr, resting mast cells; Mono, monocytes; M0, M0 macrophages; M1, M1 macrophages; M2, M2 macrophages; N, node; Neut, neutrophils; NKa, NK activated cells; NKr, resting NK cells; Plasma, plasma cells; SCC, squamous cell carcinoma; T, tumor; Tfh, follicular helper T cells; Tgd, γδT cells; Treg, regulatory T cells.

    Article Snippet: IHC and evaluationFor IHC, tissue microarray (TMA) sections were performed through deparaffinizing, rehydrating, bringing slides to a boiled sodium citrate buffer then cooling down to room temperature, blocking each section with 100–200 µL blocking solution for 2 hour at room temperature, incubating primary antibodies with PD-1 (HPA035981, 1:100; Sigma, St. Louis, MO, USA), PD-L1 (SAB4301882, 1:60; Sigma), GZMB (46890s, 1:500; Cell Signaling Technology, Danvers, MA, USA), CD68 (ab955, 1:300; Abcam, Cambridge, UK), CD56 (07-5603, 1:600; Invitrogen, Carlsbad, CA, USA), CD8a (70306s, 1:200; Cell Signaling Technology), anti-Mast cell tryptase (ab2378, 1:10,000; Abcam), CD66b (392902, 1:400; Biolegend, San Diego, CA, USA) overnight at 4°C, adding secondary antibody and staining.

    Techniques:

    Effects of CTP on CD4, CD8, IFN-γ, and IL-4 of the spleen and colorectal tumor in Apc Min/+ mice. CTP enhanced the levels of CD4, CD8, and IFN-γ, and suppressed the levels of IL-4 in (A) the spleen (400 ×, scale bar: 20 μm) and (B) colorectal tumor (200 ×, scale bar: 50 μm) analyzed by immunohistochemistry. The pixel density for the semi-quantitative densitometric analysis of protein expressions in (C) the spleen and (D) colorectal tumors were quantified. Data are presented as mean ± S.D. and analyzed via a one-way ANOVA test ( n = 3). # p

    Journal: Frontiers in Pharmacology

    Article Title: Calf Thymus Polypeptide Restrains the Growth of Colorectal Tumor via Regulating the Intestinal Microbiota-Mediated Immune Function

    doi: 10.3389/fphar.2022.898906

    Figure Lengend Snippet: Effects of CTP on CD4, CD8, IFN-γ, and IL-4 of the spleen and colorectal tumor in Apc Min/+ mice. CTP enhanced the levels of CD4, CD8, and IFN-γ, and suppressed the levels of IL-4 in (A) the spleen (400 ×, scale bar: 20 μm) and (B) colorectal tumor (200 ×, scale bar: 50 μm) analyzed by immunohistochemistry. The pixel density for the semi-quantitative densitometric analysis of protein expressions in (C) the spleen and (D) colorectal tumors were quantified. Data are presented as mean ± S.D. and analyzed via a one-way ANOVA test ( n = 3). # p

    Article Snippet: Other sections of the colorectum (especially the colorectal tumor portion) and spleen were blocked with 5% bovine serum albumin (Gen-view Scientific, Galveston, TX, United States ) for 4 h and incubated with primary antibodies including CD4 (25229S, 1:200 dilution) and CD8 (98941S, 1:800 dilution) (Cell Signaling Technology, Danvers, MA, United States ), IFN-γ (PA5-95560, 1:1000 dilution) and IL-4 (PA5-25165, 1:50 dilution) (Invitrogen, Carlsbad, CA, United States ) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (E-AB-1003 or E-AB-1001) (Elabscience, Wuhan, China) at 4°C for 2 h. Color development was performed using the Metal Enhan DAB Substrate Kit (34,065, Thermofisher, Carlsbad, CA, United States ), and hematoxylin was used for counterstaining.

    Techniques: Mouse Assay, Immunohistochemistry

    CTP regulated the expression of proteins associated with immunity in the spleen and colorectal tumors of Apc Min/+ mice. CTP regulated the expression levels of CD4, CD8, CTLA4, PD-L1, and IL-2 in (A) the colorectal tumor and (B) spleen of Apc Min/+ mice (C) CTP regulated the expression levels of Calcineurin A, NFAT1, P-PI3K, P-AKT, P-IKKα/β, and P-NF-κB p65 in the spleen of Apc Min/+ mice. Quantification data were normalized by GAPDH and their corresponding total proteins were reported as fold change with respect to the expression data from the corresponding vehicle-treated mice ( n = 4).

    Journal: Frontiers in Pharmacology

    Article Title: Calf Thymus Polypeptide Restrains the Growth of Colorectal Tumor via Regulating the Intestinal Microbiota-Mediated Immune Function

    doi: 10.3389/fphar.2022.898906

    Figure Lengend Snippet: CTP regulated the expression of proteins associated with immunity in the spleen and colorectal tumors of Apc Min/+ mice. CTP regulated the expression levels of CD4, CD8, CTLA4, PD-L1, and IL-2 in (A) the colorectal tumor and (B) spleen of Apc Min/+ mice (C) CTP regulated the expression levels of Calcineurin A, NFAT1, P-PI3K, P-AKT, P-IKKα/β, and P-NF-κB p65 in the spleen of Apc Min/+ mice. Quantification data were normalized by GAPDH and their corresponding total proteins were reported as fold change with respect to the expression data from the corresponding vehicle-treated mice ( n = 4).

    Article Snippet: Other sections of the colorectum (especially the colorectal tumor portion) and spleen were blocked with 5% bovine serum albumin (Gen-view Scientific, Galveston, TX, United States ) for 4 h and incubated with primary antibodies including CD4 (25229S, 1:200 dilution) and CD8 (98941S, 1:800 dilution) (Cell Signaling Technology, Danvers, MA, United States ), IFN-γ (PA5-95560, 1:1000 dilution) and IL-4 (PA5-25165, 1:50 dilution) (Invitrogen, Carlsbad, CA, United States ) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (E-AB-1003 or E-AB-1001) (Elabscience, Wuhan, China) at 4°C for 2 h. Color development was performed using the Metal Enhan DAB Substrate Kit (34,065, Thermofisher, Carlsbad, CA, United States ), and hematoxylin was used for counterstaining.

    Techniques: Expressing, Mouse Assay

    Validation of shRNA screen hits in H1650 and H1650-M3 cells using the clonogenic assay technique. ( A ) Clonogenic assay performed on H1650 and H1650-M3 to validate siRNA screen hits for BRCA1 , ORC5L , RFC3 , and POLS . The charts represent the percentage of viable cells 15 days after transfection with the indicated siRNA oligonucleotides compared to that of scramble-siRNA-transfected control cells. Each bar represents mean ± SD of data collected from ten fields from three different six-well plates (n = 30). ( B ) Knockdown efficiency for clonogenic assay upon transfection with the indicated siRNA oligonucleotides. Samples were collected 3 days post-transfection and analyzed by immunoblot analysis with antibodies against Total caspase 3, alpha-tubulin and Cleaved-caspase 3. Each bar represents mean ± SD of three replicates. p-value *

    Journal: eLife

    Article Title: TGF-β reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24− cancer cells

    doi: 10.7554/eLife.21615

    Figure Lengend Snippet: Validation of shRNA screen hits in H1650 and H1650-M3 cells using the clonogenic assay technique. ( A ) Clonogenic assay performed on H1650 and H1650-M3 to validate siRNA screen hits for BRCA1 , ORC5L , RFC3 , and POLS . The charts represent the percentage of viable cells 15 days after transfection with the indicated siRNA oligonucleotides compared to that of scramble-siRNA-transfected control cells. Each bar represents mean ± SD of data collected from ten fields from three different six-well plates (n = 30). ( B ) Knockdown efficiency for clonogenic assay upon transfection with the indicated siRNA oligonucleotides. Samples were collected 3 days post-transfection and analyzed by immunoblot analysis with antibodies against Total caspase 3, alpha-tubulin and Cleaved-caspase 3. Each bar represents mean ± SD of three replicates. p-value *

    Article Snippet: The following antibodies were used for immunofluorescence: Anti-gamma H2A.X (phospho S139) antibody (Abcam, Cambridge, MA); cat. # ab11174 (RRID: AB_297813 ) Anti-53BP1 antibody (Abcam, Cambridge, MA); cat. # ab36823 (RRID: AB_722497 ) Alexa Fluor 488 donkey-anti-rabbit IgG (H+L) (Invitrogen); cat # A21206 Alexa Fluor 568 donkey-anti-mouse IgG (H+L) (Invitrogen); cat # 10037 The following antibodies were used for immunoblot analysis: Cleaved caspase-3 (Asp 175) antibody (Cell Signaling Technology, Danvers, MA); cat. # 9661 (RRID: AB_2341188 ) Anti-alpha-tubulin antibody (Millipore, Billerica, MA); cat. # MABT205 (RRID: AB_11204167 ) Anti-BLM antibody (Atlas Antibodies, Bromma, Sweden); cat. # HPA005689 (RRID: AB_1845372 ) Anti-BRCA2 antibody (Atlas Antibodies); cat. # HPA026815 (RRID: AB_10602692 ) Anti-Rad50 antibody (Millipore); cat. # 05–525 (RRID: AB_309782 ) Anti-Ras-GAP antibody (BD Biosciences); cat. # 610040 (RRID: AB_397455 ) Anti-RDM1 antibody (Sigma-Aldrich, St. Louis, MO); cat. # HPA024794 (RRID: AB_1856039 ) Anti-WRN antibody (Cell Signaling Technology, Danvers, MA); cat. # 4666 (RRID: AB_10692114 )

    Techniques: shRNA, Clonogenic Assay, Transfection