primary antibodies against ccl5  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: CCL5 immunohistochemical staining in TCs and ICs. Upper row, ( A ) TCs and ICs, CCL5 negative, ( B ) TCs CCL5 positive with IRS = 8 (ICs, negative); lower row, ( C ) ICs CCL5 positive with 20% positivity (TCs, negative). All the images are at 40× magnification, and the scale bar represents 20 µm.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: K–M analysis: association of CCL5 staining in TCs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: K–M analysis: association between CCL5 expression in TCs and prognosis. Positive CCL5 staining in TCs was associated with a shorter mean OS ( p = 0.020), mean DSS ( p = 0.011), and mean RFS ( p = 0.032) than negative CCL5 staining.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses: Association of CCL5 staining in TCs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: K–M analysis: association of CCL5 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: K–M analysis: association between CCL5 expression in ICs and prognosis. Positive CCL5 staining in ICs was associated with a longer mean OS ( p = 0.029), mean DSS ( p = 0.003), and mean RFS ( p = 0.002) than negative CCL5 staining.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses: association of CCL5 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: K–M analysis: association of chemotherapy stratified by CCL5 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: K–M analysis: association of chemotherapy with prognosis (OS, DSS, and RFS) in the tumor stage 3 + 4 group stratified by negative CCL5 staining in the IC. In the tumor stage 3 + 4 group with negative CCL5 IC staining, chemotherapy treatment was associated with longer OS ( p = 0.004), longer DSS ( p = 0.011) and longer RFS ( p = 0.024).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients

doi: 10.3390/ijms25126325

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses: association of chemotherapy stratified by CCL5 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE buffer (pH 9) and peroxidase blocking (Dako, Hamburg, Germany), primary antibodies against CCL5 (rabbit polyclonal RANTES antibody, Cat. No. ab9679, dilution 1:200; Abcam, Cambridge, UK) were applied for 30 min.

Techniques: Staining

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: The overall depiction of differential expression levels of CCL5 in ccRCC and normal tissues, and its correlation with clinicopathological characteristics and outcomes in TCGA and FUSCC cohorts. ( A ) Representative immunohistochemistry images detecting CCL5 expression in paired ccRCC and normal tissues from FUSCC cohort (n=20). ( B ) Cumulative results of CCL5 IHC score from FUSCC cohort using the Students' t test. ( C ) Differential CCL5 expression in 533 tumor and 72 adjacent normal kidney samples from TCGA cohort using the Students' t test. ( D ) Distribution of paired Tumor/Normal ratio using RT-qPCR analysis from FUSCC cohort. ( E ) Western blotting assay of CCL5 expression in 4 paired fresh ccRCC and para-tumor kidney tissues. ( F ) Differential CCL5 expression in paired tumor, normal and metastatic lymph node (mlymph node) using the Students' t test (n=17). ( G ) Differential CCL5 mRNA expression with clinical AJCC stage and pathological WHO/ISUP grade using one-way ANOV test. ( H ) Multivariate Cox regression analysis enrolling clinicopathological indicators and T/N ratio in predicting overall survival displayed in a forest plot.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Quantitative Proteomics, Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Prognostic implications of CCL5 expression patterns in large-scale patients with ccRCC from the FUSCC and TCGA cohorts. ( A-B ) Kaplan-Meier and log-rank tests identify predictive value of CCL5 expression in prognostic prognosis in discovery set (n=290) from FUSCC cohort. ( C-D ) Kaplan-Meier and log-rank tests identify prognostic value of CCL5 expression in testing set (n=102) from FUSCC cohort. ( E-F ) Kaplan-Meier and log-rank tests identify prognostic value of CCL5 expression in validation set (n=530) from TCGA cohort.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Expressing, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Inhibition of CCL5 restrains proliferation, migration capacities of ccRCC cells and chemotaxis of macrophages. ( A ) Western blotting assay of CCL5 expression in transfected A498 and 786O cell lines. The data represent three independent experiments. ( B ) Cell viability of negative control and transfected ccRCC cells using CCK-8 assay. ( C ) Effect of CCL5-siRNA transfection on cell migration was determined using wound-healing assay. Histograms and Students's t test were used to evaluate the significance between two groups. ( D ) Correlation of CCL5 and epithelial-mesenchymal transition (EMT)-related markers in 533 patients with ccRCC from TCGA were detected by Spearson's correlation analysis. ( E ) The linear relationship of CCL5 expression with PI3K/AKT signaling pathway and tumor proliferation signature by Spearson's correlation. ( F ) Using Western blotting assay, expression of crucial markers in the EMT and PI3K/AKT signaling pathways were assessed.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Inhibition, Migration, Chemotaxis Assay, Western Blot, Expressing, Transfection, Negative Control, CCK-8 Assay, Wound Healing Assay, Protein-Protein interactions

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Inhibition of CCL5 restrains proliferation and chemotaxis of THP1-derived TAMs. ( A ) THP1 cells were induced differentiation into macrophages (M0) using PMA treatment and further transformed into M2 phenotype macrophages (specifically THP1-derived TAMs) induced by IL-4. ( B ) Using Western blotting assay, expression of iNOS, Arg-1, TGF-β, and CCL5 were assessed in PMA induced THP1 cells and THP1-derived TAMs. ( C ) CCK8 assay was supplemented to explore the effect of CCL5 on the proliferation ability of THP1-derived TAMs. ( D ) Representative macrophage migration images of the control group, conditioned medium (CM) of tumor cells group and the CM of CCL5-knockdown ccRCC cells. Histograms and Students's t test were used to evaluate the significance between two groups.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Inhibition, Chemotaxis Assay, Derivative Assay, Transformation Assay, Western Blot, Expressing, CCK-8 Assay, Migration, Control, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Perturbation of CCL5 in immune microenvironment of ccRCC and its distribution on TAMs. ( A ) Differential CCL5 expression distinguished the individual with ccRCC into two immunophenotypes using unsupervised clustering xCell algorithm for patients with ccRCC from TCGA database. ( B ) Cumulative results showing subpopulations gated on CCL5 high CD45 + leucocytes from fresh human ccRCC samples (n=20). ( C ) Representative immunohistochemistry images of CCL5 expressed macrophages. ( D ) Co-labeled with macrophage marker CD68 (red) and CCL5 (green) in ccRCC tissues. Nuclei were counterstained blue with DAPI. The right panel was merged by the aforementioned three images. ( E ) Opal multiplex immunofluorescence (mIF) was implemented to determine presence of tertiary lymphoid structures (TLS), abundance of tumor associated lymphocytes (TILs) and other cells, and PD-L1 expression in accordance to CCL5 expression on a multispectral imaging system in ccRCC tissues. ( F ) Histogram showing the aggregation of TILs and presence of TLS in ccRCC samples with different CCL5 expression levels. The bar graphs means the number of cases with TILs aggregation, immature TLS or mature TLS. The percentages written on top of each group means the proportion of this group of samples in the CCL5high or CCL5high group. ( G ) Histogram showing the percent of CD68 + CD163 + macrophage M2 in stromal and tumor samples with different CCL5 expression levels using unpaired t test. ( H ) Histogram showing the percent of PD-L1 + macrophages in ccRCC samples with different CCL5 expression levels using unpaired t test.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Expressing, Immunohistochemistry, Labeling, Marker, Multiplex Assay, Immunofluorescence, Imaging

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Intra-tumoral CCL5+ TAMs exposed a distinct subset with pro-tumorigenic exhausted features and impaired total CD8 + T cell function of ccRCC ( A ) We next evaluated the Relationship between transcriptomic CCL5 expression and cells infiltration level in the TME among 33 cancers in the TCGA database using Spearson's correlation analysis. ( B-C ) Representative images and histogram of flow cytometry showed infiltration of CCL5 high CD68 + cells in total CD45 + CD68 + cells in tumor and peritumor tissues (n=15). Data were analyzed using the Students' t test. ( D ) Differential level of immune checkpoints activation, exhaustion signatures and CXCL13 expression were compared based on Cibersort algorithm between CCL5 high and CCL5 low ccRCC samples from TCGA cohort using unpaired t test. ( E ) Differential proliferation biomarker Ki-67 expression in CD8 + T cells within CCL5 + or CCL5 - TAMs ccRCC fresh tumor tissues using flow cytometry analysis and unpaired t test. ( F ) Differential immunity stimulatory effectors in CD8 + T cells within CCL5 + or CCL5 - TAMs ccRCC fresh tumor tissues using flow cytometry analysis and unpaired t test. ( G ) Differential immune checkpoints molecules within CCL5 + or CCL5 - TAMs ccRCC fresh tumor tissues using flow cytometry analysis and unpaired t test.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Cell Function Assay, Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Accumulation of CCL5 + TAMs distinguished clinical outcomes in patients with ccRCC. ( A ) The necessary transcription signatures composing CCL5 + TAMs were enrolled in Lasso regression analysis to establish the CCL5 + TAMs model for 530 patients with ccRCC from validation TCGA cohort. ( B-C ) Kaplan-Meier and log-rank tests identify prognostic value of TAMs infiltration alone in validation set (n=530) from TCGA cohort. ( D-E ) Kaplan-Meier and log-rank tests identify prognostic value of CCL5 + TAMs infiltration in validation set (n=530) from TCGA cohort.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Biomarker Discovery

Journal: Frontiers in Pharmacology

Article Title: Biejiajian Pill Promotes the Infiltration of CD8 + T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5

doi: 10.3389/fphar.2021.771046

Figure Lengend Snippet: BJJP enhances the expression of CCL5 in vivo . (A) Relative mRNA levels of CCL5, CCL9, CCL17, CXCL9, CXCL10 and CXCL12 . The expression of CCL5 in tumor tissues was examined using (B) immunofluorescence and (C) Western blotting. (D) Levels of CCL5 in mice serum were assessed by ELISA. The mean ± SD of three independent experiments is shown for each sample. * p < 0.05, ** p < 0.01 vs. controls; # p < 0.05 vs. CCL5-Ctrls.

Article Snippet: Afterward, the membranes were probed with specific primary antibodies against CCL5 (1:1,000; Bioss bsm-54125R) and GAPDH (1:1,000; Servicebio GB11002), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, In Vivo, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Biejiajian Pill Promotes the Infiltration of CD8 + T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5

doi: 10.3389/fphar.2021.771046

Figure Lengend Snippet: BJJP enhances the expression of CCL5 in vitro . H 22 cells were exposed to designated concentrations of BJJP (L, H) for 24 h. (A,B) Increased mRNA and protein levels of CCL5, respectively, after treatment with BJJP for 24 h. (C) A schematic diagram of the spleen cell migration model. (D) Migrating CD8 + T cells were detected by flow cytometry. The data represents the mean ± SD of technical triplicates. * p < 0.05, ** p < 0.01 vs. controls.

Article Snippet: Afterward, the membranes were probed with specific primary antibodies against CCL5 (1:1,000; Bioss bsm-54125R) and GAPDH (1:1,000; Servicebio GB11002), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, In Vitro, Migration, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Biejiajian Pill Promotes the Infiltration of CD8 + T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5

doi: 10.3389/fphar.2021.771046

Figure Lengend Snippet: Inhibiting the expression of CCL5 reverses the effects of BJJP. (A) Western blotting was used to detect the relative expression of CCL5 in H 22 cells transfected with shRNA-CCL5 or shRNA-Ctrl. The data is presented as the mean ± SD from three independent experiments. H 22 cells in which CCL5 was knocked down were injected subcutaneously in BALB/c mice. The subcutaneous tumors obtained from the mice were then (B) photographed, (C) measured and (D) weighed. (E,F) The percentage of CD8 + T cells after tumor digestion was obtained using flow cytometry. Data are presented as the mean ± SD ( n = 6). (A) ** p < 0.01 vs. controls. (C,D) * p < 0.05, ** p < 0.01 vs controls; ## p < 0.01 vs. CCL5-Ctrls; Δ p < 0.05, ΔΔ p < 0.01 vs. BALB/c mice BJJP.

Article Snippet: Afterward, the membranes were probed with specific primary antibodies against CCL5 (1:1,000; Bioss bsm-54125R) and GAPDH (1:1,000; Servicebio GB11002), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, Western Blot, Transfection, shRNA, Injection, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Biejiajian Pill Promotes the Infiltration of CD8 + T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5

doi: 10.3389/fphar.2021.771046

Figure Lengend Snippet: BJJP regulates CCL5 in human HCC cells. The HepG2 and SMMC-7721 human hepatocellular carcinoma cells were exposed to designated concentrations of BJJP (L, H) for 24 h. (A,B) Increased mRNA and protein levels of CCL5, respectively, after treatment with BJJP for 24 h. (C) The distribution of CCL5 fluorescence in HepG2 and SMMC-7721 cells was examined using a fluorescence microscope (scale bar, 100 μm, 200×). The mean ± SD of three independent experiments is shown for each sample. * p < 0.05, ** p < 0.01 vs. controls.

Article Snippet: Afterward, the membranes were probed with specific primary antibodies against CCL5 (1:1,000; Bioss bsm-54125R) and GAPDH (1:1,000; Servicebio GB11002), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Fluorescence, Microscopy

Journal: Cell death & disease

Article Title: CCL5 derived from tumor-associated macrophages promotes prostate cancer stem cells and metastasis via activating β-catenin/STAT3 signaling.

doi: 10.1038/s41419-020-2435-y

Figure Lengend Snippet: Fig. 1 TAMs-derived CCL5 was elevated in prostate cancer and associated with metastasis. a, b The mRNA and protein expression differences of indicated genes between prostate cancer tumor tissues and para-carcinoma tissues (n = 3). c Elisa assay indicated that the blood samples of prostate cancer patients (n = 30) exhibited elevated expression of CCL5 when compared with that of healthy male participants (n = 30). d Prostate cancer patients with higher Gleason grade exhibited an increased CCL5 accumulation in the blood (n = 30). e Tissue immunofluorescence assay suggested that CCL5 and the macrophage marker CD163 were co-localized in both the primary tumor and metastatic lymph node of prostate cancer patients. Scale bar, 10 μm. f TAMs exhibited increased secretion of CCL5 than immature macrophages or prostate cancer cells. CCL5 concentrations in the cell culture supernatants of THP1-derived macrophages (M0), THP1-derived TAMs (M2), DU145 and PC3 cells were measured using Elisa method (n = 10). All values are presented as the mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: Briefly, the expression levels of CCL5, STAT3, and ALDH1A1 in prostate cancer tissue specimens were determined by the IHC method using primary antibodies against CCL5 (AF5151, Affinity), STAT3 (60199-1-lg, Proteintech) and ALDH1A1 (BF0220, Affinity).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Cell Culture

Journal: Theranostics

Article Title: Minimally invasive implantation and decreased inflammation reduce osteoinduction of biomaterial

doi: 10.7150/thno.39507

Figure Lengend Snippet: T cell-mediated adaptive immune response recruits MSCs. ( A ) Activation of NF-κB pathway in T cells after TNF-α stimulation was detected by Western Blot. ( B ) Western Blot showed the secretion of CCL5 by T cells after the NF-κB pathway was activated by TNF-α or blocked by PDTC. ( C ) Transwell image showed the chemotaxis of T cells to MSCs after the NF-κB pathway was activated by TNF-α or blocked by PDTC. ( D ) Quantitative comparison of the chemotaxis of T cells to MSCs in each group. (n=6) *** P < 0.001, NS, no statistical significance. ( E ) TNF-α activates the NF-κB pathway to increase the release of CCL5 in T cells.

Article Snippet: The membranes were soaked in 5% non-fat milk for 1 h and incubated overnight with primary antibodies against CCL5 (1:1000, ABclonal, Wuhan, China), NF-κB p65 (1:1000, CST, U.S.A.), phospho-NF-κB p65 (1:1000, CST, U.S.A.), or GAPDH (1:5000, Proteintech, U.S.A.) at 4°C.

Techniques: Activation Assay, Western Blot, Chemotaxis Assay, Comparison