Journal: The Journal of biological chemistry

Article Title: Ligand exchange during unfolding of cytochrome c.

doi: 10.1074/jbc.274.25.17853

Figure Lengend Snippet: FIG. 1. A and B, equilibrium titration curves for the unfolding of cyt c by GdnHCl (GHCl) at pH 5.9. In A, the populations represented by the cyan squares were determined from the intensity of the tryptophan fluorescence at 344 nm, those represented by the magenta diamonds were determined from CD measurements at 222 nm, and those repre- sented by the dark blue circles were determined from resonance Raman measurements by following the formation of HM. In B, the dark blue circles, the green squares, and the red triangles are the populations of the HM, HW, and HH forms, respectively, determined from resonance Raman measurements. The magenta line in B is the CD titration curve reproduced from A to guide the eye. C, pictorial illustration of the relative free energy changes of HM and HH with respect to HW as a function of the GdnHCl concentration. The red, green, and dark blue curves correspond to 0.0, 2.4, and 2.7 M GdnHCl concentrations, respectively.

Article Snippet: For the equilibrium studies, the protein was diluted into pH 3.0 or 5.9 acetate buffer (0.1 M) at the desired concentration of guanidine hydrochloride (GdnHCl, ultra pure grade from Pierce) a few hours prior to the experiments.

Techniques: Titration, Fluorescence, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: Ligand exchange during unfolding of cytochrome c.

doi: 10.1074/jbc.274.25.17853

Figure Lengend Snippet: FIG. 2. A and B, equilibrium titration curves for the unfolding of cyt c by GdnHCl (GHCl) at pH 3.0. In A, the populations represented by the cyan squares were determined from the intensity of the tryptophan fluorescence at 344 nm, those represented by the magenta diamonds were determined from CD measurements at 222 nm, and those repre- sented by the dark blue circles were determined from resonance Raman measurements by following the formation of HM. In B, the dark blue circles, the green squares, and the cyan diamonds are the populations of the HM, HW, and 5C forms, respectively, determined from resonance Raman measurements. The magenta line in B is the CD titration curve reproduced from A to guide the eye. C, pictorial illustration of the relative free energy changes of HM and 5C with respect to HW as a

Article Snippet: For the equilibrium studies, the protein was diluted into pH 3.0 or 5.9 acetate buffer (0.1 M) at the desired concentration of guanidine hydrochloride (GdnHCl, ultra pure grade from Pierce) a few hours prior to the experiments.

Techniques: Titration, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Ligand exchange during unfolding of cytochrome c.

doi: 10.1074/jbc.274.25.17853

Figure Lengend Snippet: FIG. 3. Time course for the populations of the various heme- ligand coordinated states during the unfolding of cyt c deter- mined from tryptophan fluorescence and resonance Raman scattering measurements. The folded protein at pH 5.9 was unfolded at pH 3.8 (A) and at pH 5.8 (B) in 4.0 M GdnHCl. The magenta crosses were determined from the intensity of the tryptophan fluorescence. The green squares, the red triangles, the dark blue circles, and the cyan diamonds are the populations of the HW, HH, HM, and 5C forms, respectively, determined from the resonance Raman measurements. The cyan wedges in B represent the intensity of the 397-cm21 Raman line that is sensitive to the structure of the heme pocket. The solid lines are fits to the data using the model presented in Scheme 2. The rate constants are listed in Table I.

Article Snippet: For the equilibrium studies, the protein was diluted into pH 3.0 or 5.9 acetate buffer (0.1 M) at the desired concentration of guanidine hydrochloride (GdnHCl, ultra pure grade from Pierce) a few hours prior to the experiments.

Techniques: Fluorescence

Journal: The Journal of biological chemistry

Article Title: Ligand exchange during unfolding of cytochrome c.

doi: 10.1074/jbc.274.25.17853

Figure Lengend Snippet: FIG. 4. Time course for the populations of the various heme- ligand coordinated states during the folding of cyt c determined from tryptophan fluorescence and resonance Raman scattering measurements. The protein was unfolded at pH 3.6 (A) and pH 5.6 (B) in 4.4 M GdnHCl and refolded at pH 5.0 to a final GdnHCl concentration of 0.7 M. The symbols are defined in the legend for Fig. 3.

Article Snippet: For the equilibrium studies, the protein was diluted into pH 3.0 or 5.9 acetate buffer (0.1 M) at the desired concentration of guanidine hydrochloride (GdnHCl, ultra pure grade from Pierce) a few hours prior to the experiments.

Techniques: Fluorescence, Concentration Assay

Journal: Heliyon

Article Title: Mapping the 5-HTergic neural pathways in perimenopausal mice and elucidating the role of oestrogen receptors in 5-HT neurotransmission

doi: 10.1016/j.heliyon.2024.e27976

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: Rabbit anti-mouse MAOA antibody , A1354 , ABclonal , 1:1000 (WB).

Techniques:

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: DLGAP5 expression levels in breast tissues of patients with BC. (A) mRNA expression levels of DLGAP5 in BC tissues compared with normal breast samples. (B) mRNA expression levels of DLGAP5 in an expanded sample size following analysis of the GSE29431 and GSE61304 datasets. (C and D) ROC curve based on DLGAP5 expression levels in (C) GSE29431 and (D) GSE61304 datasets for predicting breast cancer tissue classification. (E) PFS and (F) OS analysis of patients with BC with low and high DLGAP5 expression levels. ***P<0.001. DLGAP5, discs large-associated protein 5; PFS, progression-free survival; OS, overall survival; BC, breast cancer; ROC, receiver operating characteristic; HR, hazard ratio.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Parameters associated with DLGAP5 expression in the GOBO database in patients with breast cancer. (A) Boxplot of DLGAP5 mRNA expression levels stratified based on the molecular subtypes. Boxplot of DLGAP5 mRNA expression levels stratified by (B) PAM50 classification, (C) estrogen receptor status and (D) tumor grade. The expression levels of DLGAP5 were compared among subtypes of breast cancer. P<0.00001 vs. basal, ER-neg and grade 1. DLGAP5, discs large-associated protein 5. HER2, human epidermal growth factor receptor-2; LumA, luminal A subtype; LumB, luminal B subtype; ER, estrogen receptor; -neg, negative; -pos, positive.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Representative immunohistochemistry images of DLGAP5 expression levels in breast cancer tissue samples. (A and B) Strong, weak and null expression levels of DLGAP5 in (A) breast tumor tissue (n=160) and (B) normal breast tissue (n=32). Magnification, ×400. DLGAP5, discs large-associated protein 5.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Immunohistochemistry, Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Association of DLGAP5 expression levels with clinical characteristics of patients with breast cancer.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Underlying molecular mechanisms of DLGAP5 activity in breast cancer. (A) Different GO enriched terms associated with genes co-expressed with DLGAP5 in breast cancer. (B) KEGG pathway enrichment of the co-expressed genes. (C) Subcellular localization of DLGAP5. The green intensity represents the localization probability. (D) Domain demonstration of DLGAP5 and its LIR motif (in red) exhibiting conservation across species. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DLGAP5, discs large-associated protein 5.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Activity Assay

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Effects of DLGAP5 on MDA-MB-231 cell proliferation. (A) Expression levels of DLGAP5 in MDA-MB-231 cells transfected with si-NC, si-DLGAP5-1 and si-DLGAP5-2 was determined by reverse transcription-quantitative PCR after transfection for 48 h. (B) Cell Counting Kit-8 assay was used to detect the effects of DLGAP5 on MDA-MB-231 cell proliferation. (C and D) Flow cytometry was used to detect the effects of DLGAP5 on cell cycle regulation in MDA-MB-231 cells. *P<0.05 and ***P<0.001 vs. si-NC; **P<0.01 si-DLGAP5-1 vs. si-NC; ### P<0.001 si-DLGAP5-2 vs. si-NC. si, small interfering; NC, negative control; DLGAP5, discs large-associated protein 5.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Flow Cytometry, Negative Control

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: The primers and siRNA/shRNA sequences used in this study

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: Negative Control

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: OS tumor tissues and cell lines exhibit Tspan7 upregulation. A – D The mRNA-level expression of Tspan7 was markedly increased in OS tissues and cell lines relative to corresponding normal controls (OBs and MSCs) in the GSE (14,359, 12,865, 33,383, and 42,352) datasets. E Relative to Mg63 cells, HOS and Saos2 cells exhibited significantly increased Tspan7 mRNA levels in a qRT-PCR assay, whereas this level was decreased in U2OS cells. GAPDH served as a normalization control. Data are means ± SEM from two separate experiments. F Western blotting results showed that Tspan7 in HOS and Saos2 but not U2OS was markedly higher compared with Mg63 cell. Bands were normalized to the β-actin loading control. Data were presented as the means ± SD of two independent experiments. G ROC curves and AUC values for OS based upon the GSE33383 and GSE42352 dataset. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ROC, receiver operating characteristic; AUC area under the curve, SD standard deviation. ** P < 0.01, **** P < 0.0001. Student’s t-test

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Standard Deviation

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: Tspan7 knockdown suppresses the proliferation of OS cells; Tspan7-associated biological functions based on RNA-seq analysis. A The knockdown of Tspan7 was confirmed via qRT-PCR at 72 h post-transfection in cells transfected with siTspan7 and siNC. B The viability of cells transfected with siTspan7 or siNC was assessed via CCK-8 assay at 72 h post-transfection. C The effect of Tspan7 silencing on HOS cell growth was assessed in a colony formation assay. Statistical results of colony formation numbers normalized to the NC group were presented. D Top DEGs identified following Tspan7 knockdown in HOS cells are arranged in a heatmap, with shNC cells being used for comparison. E The 764 DEGs identified when comparing shTspan7 and shNC groups at an adjusted |log2FoldChange|≥ 1 and P < 0.05 are shown in a volcano plot, including 416 upregulated genes (red dots) and 348 downregulated genes (green dots). F GO analyses exploring the cellular components, molecular functions, and biological processes in which DEGs were enriched were plotted based upon gene number, with darker blue dots indicating more significant enrichment. G KEGG enrichment signaling analysis results. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Student’s t-test. NC negative control, si small interfering RNA, DEGs different expression genes, GO gene ontology, KEGG kyoto encyclopedia of genes and genomes

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: Knockdown, RNA Sequencing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Comparison, Negative Control, Small Interfering RNA, Expressing

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: Tspan7 contributes to OS cell metastasis in vitro. A Cells in which Tspan7 was stably knocked down using the indicated shRNA constructs exhibited reduced Tspan7 mRNA levels and protein levels as compared to the shNC group in qRT-PCR (left panel) and western blotting (right panel) assays. B Cells were imaged at 10× magnification, with successfully infected cells exhibiting GFP expression, demonstrating near 100% transduction efficiency. C , D The effect of Tspan7 downregulation on HOS and Saos2 cell migration was assessed by wound healing and Transwell assays. E Matrigel-coated invasion assays were used to assess the invasivity of HOS and Saos2 cells stably expressing shTspan7 or shNC. F Fluorescence microscopy (upper panel), qRT-PCR (lower left panel), and western blotting (lower right panel) were used to gauge the degree of Tspan7 overexpression in OE-Tspan7 and mock U2OS cells. G Wound healing assays were conducted using U2OS cells stably transfected with OE-Tspan7 or mock constructs. H U2OS cells stably transfected with OE-Tspan7 or mock constructs were utilized in Transwell migration assays. I Matrigel-coated invasion assays were used to assess the invasivity of U2OS cells stably expressing OE-Tspan7 or mock constructs. Representative images are shown, and all migratory and invasive cells were counted. Experiments were repeated in triplicate, and data are means ± SD. shRNA, short hairpin RNA; GFP green fluorescence protein. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Student’s t-test

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: In Vitro, Stable Transfection, shRNA, Construct, Quantitative RT-PCR, Western Blot, Infection, Expressing, Transduction, Migration, Fluorescence, Microscopy, Over Expression, Transfection

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: Tspan7 controls EMT induction, and knocking down of it suppresses in vivo OS metastasis. A Expression of EMT markers in HOS and U2OS cells stably knockdown or overexpression Tspan7. B Quantification of the western blotting results presented in A . C Representative images of lungs harboring OS metastases (arrows) on day 30 following the injection of HOS cells expressing shNC or shTspan7. D The presence of metastatic foci (dotted lines and black arrows) in lung pathological sections was assessed after H&E staining. Blue arrows indicate alveolar tissue. Scale bar = 40 μm and 10 μm. E Numbers of lung metastases per group were quantified (5 mice/group). F The administration of shTspan7 did not influence the body weights in the mice. G The administration of shTspan7 reduced slightly the lung weights in the mice. * P < 0.05, ** P < 0.01. Student’s t-test

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: In Vivo, Expressing, Stable Transfection, Knockdown, Over Expression, Western Blot, Injection, Staining

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: Tspan7 complexes with β1 integrin to promote FAK-Src-Ras-ERK1/2 pathway activation. A Co-immunoprecipitation assays revealed interactions between Tspan7 and integrin β1. B Integrin β1 levels were assessed in HOS-shTspan7 and U2OS-Tspan7 cells via western blotting. C Proteins associated with the FAK-Src-Ras-ERK1/2 pathway (FAK, pFAK Y397 , pFAK Y925 , Ras, ERK1/2, and pERK1/2) were assessed by western blotting, with β-actin as a loading control. D Quantification of the western blotting results presented in C . E Signaling downstream of Ras in Tspan7-overexpressing U2OS cells treated with Salirasib (50 μM) was assessed via western blotting. F U2OS-Tspan7 cells treated with or without Salirasib (50 μM) were assessed by migration and invasion assays. Representative cell images are shown, and cells were counted. All experiments were repeated two or three times. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Control, Migration

Journal: Cancer Cell International

Article Title: Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

doi: 10.1186/s12935-022-02591-1

Figure Lengend Snippet: A schematic mechanism of Tspan7 in osteosarcoma progression. Tspan7 interacts with integrin β1 to promote osteosarcoma metastasis through activating integrin-mediated FAK-Src-Ras-ERK1/2 signaling transduction and enhancing EMT process of osteosarcoma cells

Article Snippet: Blots were blocked with 5% non-fat milk in TBST, and were then incubated overnight at 4 °C with the antibodies specific for the following: Tspan7 (1:300, #A13555, ABclonal), FN1 (1:1000, #A12932, ABclonal), N-Cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), Slug (1:1000, #9585, CST), Snai1 (1:1000, #3879, CST), phospho-FAK Y397 (1:500, #ab81298, Abcam), phospho-FAK Y925 (1:500, #3284 T, CST), total-FAK (1:1000, #ab40794, Abcam), phospho-Src Y529 (1:500, # AP0185, ABclonal), total-Src (1:500, #A19119, ABclonal), Ras (1:1000, #ab52939, Abcam), phospho-ERK1/2 (1:1000, #4370, CST), total-ERK1/2 (1:1000, #4695, CST), integrin β1/ITGB1 (1:500, #A2217, ABclonal), Flag (1:1000, #F7425, Sigma), and β-actin (1:1000, #A5441, Sigma-Aldrich).

Techniques: Transduction