rabbit anti ptch1 polyclonal antibody  (Novus Biologicals)

Journal: Nature Communications

Article Title: TSPAN8 promotes cancer cell stemness via activation of sonic Hedgehog signaling

doi: 10.1038/s41467-019-10739-3

Figure Lengend Snippet: TSPAN8 stabilizes the expression of PTCH1 by recruiting ATXN3. a MCF7 cell lysates were immunoprecipitated with control rabbit IgG or anti-TSPAN8 and immunoblotted with anti-SHH or anti-PTCH1 antibodies. Ten percent of the cell extracts was loaded as an input. b GST pulldown assay was performed by mixing purified GST-TSPAN8 or GST with the lysates of HEK293T cell expressing His-SHH or His-PTCH1-CTD (C-terminal domain of PTCH1), then immunoblotting analyses were performed (top panel). Coomassie blue staining of GST-TSPAN8 and GST was performed (bottom panel). c Immunofluorescence analysis of MCF7 cells with or without TSPAN8 overexpression was performed. DAPI was used to stain the nuclei. Scale bar = 30 μm. d MCF7 cells with or without TSPAN8 overexpression were treated with CHX (100 μg/ml) for different periods of time. Quantitation was done by densitometry with Image J Software. SHH and PTCH1 band intensities were normalized according to the levels of GAPDH, SHH, and PTCH1 in the CHX-untreated cells ( n = 3 per group, right). e MCF7 cells transfected with control siRNA or TSPAN8 siRNA were treated with 100 μg/ml CHX for the indicated periods of time then immunoblotting analyses were performed with the indicated antibodies. f MCF7 cells transfected control siRNA or TSPAN8 siRNA followed by treatment with or without MG132 (20 μM, 8 h). Immunoblotting analyses were performed with the indicated antibodies. g A vector expressing Flag- ATXN3 was co-transfected with a vector expressing GFP or GFP- TSPAN8 in HEK293T cells. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. h HEK293T cells were transfected with control siRNA or TSPAN8 siRNA together with a Flag- ATXN3 plasmid. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. i , j MCF7 cells expressing Myc- ATXN3 ( i ) or ATXN3 shRNA ( j ) were treated with MG132 (20 μM for 8 h) and SHH (100 ng/ml for 6 h). The whole-cell lysate was subjected to pulldown with His beads. All panels are representative results from three or more independent experiments

Article Snippet: The following antibodies were used in the IHC experiment: anti-TSPAN8 rabbit polyclonal antibody (1:100, Abcam, ab70007), anti-ALDHA1 rabbit polyclonal antibody (1:200, Proteintech, 15910-1-AP), anti-GLI1 rabbit polyclonal antibody (1:200, Proteintech), anti-PTCH1 rabbit polyclonal antibody (1:100, Proteintech, 17520-1-AP), anti-SHH rabbit polyclonal antibody (1:50, Proteintech, 20697-1-AP), anti-SOX2 rabbit polyclonal antibody (1:100, Proteintech, 11064-1-AP), and anti-Ki67 rabbit polyclonal antibody (1:200, Abcam, ab15580).

Techniques: Expressing, Immunoprecipitation, Control, GST Pulldown Assay, Purification, Western Blot, Staining, Immunofluorescence, Over Expression, Quantitation Assay, Software, Transfection, Plasmid Preparation, shRNA

Journal: Nature Communications

Article Title: TSPAN8 promotes cancer cell stemness via activation of sonic Hedgehog signaling

doi: 10.1038/s41467-019-10739-3

Figure Lengend Snippet: TSPAN8 promotes SHH-induced SMO phosphorylation. a , b Flag-PTCH1 ( a ) or Flag-SMO ( b ) was immunoprecipitated from MCF7 cells with or without TSPAN8 overexpression in the presence or absence of ATXN3 shRNA. Cells were treated with 100 ng/ml SHH for 6 h. Immunoblotting analyses were performed with the indicated antibodies. PLVX, lentiviral stable transfection plasmid pLVX-HA-IRES-Puro. c Endogenous SMO was immunoprecipitated from MCF7 cells with or without TSPAN8 overexpression in the presence or absence of ATXN3 shRNA. Cells were treated with 100 ng/ml SHH for 6 h. Immunoblotting analyses were performed with the indicated antibodies. P-Ser, phosphorylated serine. d MCF7 cells with or without overexpressing ATXN3 or TSPAN8 or MCF7 cells with overexpressing TSPAN8 and ATXN3 shRNA were left untreated or treated with 100 ng/ml SHH. Immunofluorescent studies were performed with the indicated antibodies. e MCF7 cells with or without overexpressing ATXN3 or TSPAN8 or MCF7 cells with overexpressing TSPAN8 and ATXN3 shRNA were transfected with a GLI1 promoter–luciferase reporter plasmid. These cells were left untreated (left) or treated (right) with 100 ng/ml SHH. Luciferase activity was measured and normalized to Renilla luciferase activity ( n = 3 per group). Two-tailed unpaired Student’s t test was performed. * P < 0.05, ** P < 0.01, and *** P < 0.001. f , g MCF7 cells with or without expressing GFP- TSPAN8 in the presence or absence of ATXN3 siRNA expression were treated with 20 μM Paroxetine hydrochloride ( f ) or 2 μM GSK180736A ( g ) together with 100 ng/ml SHH for 6 h. Cellular extracts were immunoprecipitated with anti-SMO antibody, then immunoblotting analyses tested the serine-phosphorylation expression level. h Histograms show the mean numbers and diameters of spheres formed by MCF7 cells expressing the indicated proteins and ATXN3 shRNA. i MCF7 cells expressing the indicated proteins and ATXN3 shRNA were treated with ADR (left) or PTX (right) with the different concentrations for 24 h. The cell viabilities were determined. Cell numbers with no drug was used as control ( n = 3 per group). Two-tailed unpaired Student’s t test was performed. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The following antibodies were used in the IHC experiment: anti-TSPAN8 rabbit polyclonal antibody (1:100, Abcam, ab70007), anti-ALDHA1 rabbit polyclonal antibody (1:200, Proteintech, 15910-1-AP), anti-GLI1 rabbit polyclonal antibody (1:200, Proteintech), anti-PTCH1 rabbit polyclonal antibody (1:100, Proteintech, 17520-1-AP), anti-SHH rabbit polyclonal antibody (1:50, Proteintech, 20697-1-AP), anti-SOX2 rabbit polyclonal antibody (1:100, Proteintech, 11064-1-AP), and anti-Ki67 rabbit polyclonal antibody (1:200, Abcam, ab15580).

Techniques: Phospho-proteomics, Immunoprecipitation, Over Expression, shRNA, Western Blot, Stable Transfection, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Two Tailed Test, Expressing, Control

Journal: Nature Communications

Article Title: TSPAN8 promotes cancer cell stemness via activation of sonic Hedgehog signaling

doi: 10.1038/s41467-019-10739-3

Figure Lengend Snippet: TSPAN8 correlates with PTCH1, SHH, and ATXN3 expression. a Representative IHC images of TSPAN8 in breast tumors ( n = 20) and their matched adjacent tissues. b Representative IHC images of the expression levels of SHH, PTCH1, and ATXN3 in breast tumors. c – f Positive correlation of TSPAN8 expression with SHH ( c ), PTCH1 ( d ), and ATXN3 ( e ) expression and positive correlation between PTCH1 and ATXN3 expression ( f ) are shown. * P < 0.05, ** P < 0.01, *** P < 0.001. The P values and correlation coefficient were analyzed as indicated. g Kaplan–Meier plot of survival from 90 breast cancer patients is shown. A log-rank test was used to calculate the difference between the two groups. h A mechanism of TSPAN8-enhanced Sonic Hedgehog signaling pathway. The interaction between TSPAN8 and PTCH1 leads to recruitment of ATXN3 deubiquitinating enzyme to the SHH-PTCH1 complex and subsequent deubiquitylation of PTCH1 and inhibition of proteasome-mediated degradation of SHH and PTCH1. Stabilized SHH and PTCH1 promote the binding of protein kinase GRK2 to SMO, phosphorylation and translocation of SMO to cilia, GLI1 activation for downstream gene expression, cancer cell stemness, as well as tumor formation in mice

Article Snippet: The following antibodies were used in the IHC experiment: anti-TSPAN8 rabbit polyclonal antibody (1:100, Abcam, ab70007), anti-ALDHA1 rabbit polyclonal antibody (1:200, Proteintech, 15910-1-AP), anti-GLI1 rabbit polyclonal antibody (1:200, Proteintech), anti-PTCH1 rabbit polyclonal antibody (1:100, Proteintech, 17520-1-AP), anti-SHH rabbit polyclonal antibody (1:50, Proteintech, 20697-1-AP), anti-SOX2 rabbit polyclonal antibody (1:100, Proteintech, 11064-1-AP), and anti-Ki67 rabbit polyclonal antibody (1:200, Abcam, ab15580).

Techniques: Expressing, Inhibition, Binding Assay, Phospho-proteomics, Translocation Assay, Activation Assay, Gene Expression