rabbit polyclonal tff2  (Proteintech)

Journal: Cell Death & Disease

Article Title: Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids

doi: 10.1038/s41419-024-06680-z

Figure Lengend Snippet: A Light microscopy images of colon normal organoids cultured in PROL, DIFF, or B + D media for 72 h (patient #110). Scale bar, 500 μm. B RT-qPCR analysis of the RNA levels of stemness, enterocytic and mucosecretory genes in organoids from patients #47, #86, #110, #130 for 48 h and #47, #110, #130, #159 for 72 h cultured in PROL (black), DIFF (red) or B + D (green) media. * P < 0.05, ** P < 0.01, *** P < 0.001. C Immunofluorescence analysis of the expression of enterocytic and mucosecretory protein markers (green) in organoids cultured for 48 h in PROL or B + D media (patient #158). Scale bars, 30 µm. Box-plots show the quantification of the fluorescence intensity (Log 2 MGV) (patients #92, #158 and #166; the number of organoids analyzed was 55 for KLF4, 80 for KRT19, 83 for AGR2 and 79 for TFF2). ** P < 0.01, *** P < 0.001. MGV: mean gray value.

Article Snippet: Whole-cell extracts (15–30 μg or 80 μg the case of phospho-SMAD analyses) were separated by SDS-PAGE, transferred to PVDF membranes and incubated with the following primary antibodies: rabbit polyclonal-CA1 (CUSABIO, TX, USA, #CSBPA004364GA01HU), mouse monoclonal-GAPDH (Abcam, Cambridge, UK, #ab8245), rabbit monoclonal-PTK7 (Cell Signalling, MA, USA, #25618), rabbit polyclonal-SMAD1/5/8 (Santa Cruz, TX, USA, #sc6031-R), rabbit monclonal-Phospho-SMAD1/5/8 (Cell Signalling, #9511 S) and rabbit polyclonal-TFF2 (Proteintech, IL, USA, #13681-1-AP).

Techniques: Light Microscopy, Cell Culture, Quantitative RT-PCR, Immunofluorescence, Expressing, Fluorescence

Journal: Cell Death & Disease

Article Title: Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids

doi: 10.1038/s41419-024-06680-z

Figure Lengend Snippet: A Light microscopy images of organoids (patient #110) cultured for 72 h in DIFF, BMP4, DBZ or B + D media in the absence (vehicle) or presence of calcitriol (100 nM). Scale bar, 500 μm. B RT-qPCR analysis of the RNA levels of enterocytic ( KRT20 , CA1 , KLF4 and FABP2 ) and mucosecretory ( AGR2 , TFF2 and TFF3 ) genes in organoids from patients #47, #86, #110, #130 for 48 h and #47, #110, #130, #159 for 72 h cultured in DIFF (red), BMP4 (blue), DBZ (orange) or B + D (green) media in the absence (vehicle) or presence of calcitriol (100 nM). * P < 0.05, ** P < 0.01, *** P < 0.001. C RT-qPCR analysis of the RNA levels of stemness genes in organoids from the same patients and in the same conditions as in B. D Western blot analysis and quantification of the effect of calcitriol on the expression of enterocytic (CA1) and mucosecretory (TFF2) marker proteins in organoids from four patients incubated in PROL or B + D media in the absence (vehicle) or presence of calcitriol (100 nM) for 48 h. The stemness PTK7 protein was used as control of differentiation and GAPDH as loading control. * P < 0.05. E Immunofluorescence analysis of the expression of enterocytic and mucosecretory protein markers in organoids (patient #158) cultured for 48 h in PROL or B + D media in the absence (vehicle) or presence of calcitriol (100 nM). Scale bars, 30 µm. Box-plots show the quantification of fluorescence intensity (Log 2 MGV) (patients #92, #158 and #166; the number of organoids analyzed was 68 for KLF4, 104 for KRT19, 110 for AGR2 and 79 for TFF2). ** P < 0.01, *** P < 0.001. F Western blot analysis and quantification of the level of phospho(P)-SMAD1/5/8 in organoid cultures that were incubated for 24 h with calcitriol (100 nM) or vehicle before incubation in DIFF medium or BMP4 medium (50 ng/mL) during the indicated times. MGV: mean gray value.

Article Snippet: Whole-cell extracts (15–30 μg or 80 μg the case of phospho-SMAD analyses) were separated by SDS-PAGE, transferred to PVDF membranes and incubated with the following primary antibodies: rabbit polyclonal-CA1 (CUSABIO, TX, USA, #CSBPA004364GA01HU), mouse monoclonal-GAPDH (Abcam, Cambridge, UK, #ab8245), rabbit monoclonal-PTK7 (Cell Signalling, MA, USA, #25618), rabbit polyclonal-SMAD1/5/8 (Santa Cruz, TX, USA, #sc6031-R), rabbit monclonal-Phospho-SMAD1/5/8 (Cell Signalling, #9511 S) and rabbit polyclonal-TFF2 (Proteintech, IL, USA, #13681-1-AP).

Techniques: Light Microscopy, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Marker, Incubation, Control, Immunofluorescence, Fluorescence

Journal: Bioscience Reports

Article Title: Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice

doi: 10.1042/BSR20170695

Figure Lengend Snippet: Primer sequences for qRT-PCR

Article Snippet: The diluted primary antibody was added: rabbit antimouse polyclonal antibody SIRT1 (bs-1921R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse monoclonal antibody Bax (ab32503, Abcam Inc., Cambridge, MA, U.S.A.), rabbit antimouse polyclonal antibody Bcl-2 (bs-20351R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse polyclonal antibody p53 (bs-8687R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9485, Abcam Inc., Cambridge, MA, U.S.A.) (1:500) for overnight incubation and a wash with PBS three times at room temperature, each time for 5 min. Rabbit antimouse polyclonal antibody IgG labeled by horseradish peroxidase (HRP) was added and the tissues were shaken and incubated for 1 h at 37°C, washed with PBS three times, with 5 min intervals.

Techniques:

Journal: Bioscience Reports

Article Title: Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice

doi: 10.1042/BSR20170695

Figure Lengend Snippet: ( A ) Comparison of SIRT1 protein expression of lens tissue in the normal and model groups by immunohistochemistry; ( B ) comparison of the positive expression rates of SIRT1 protein in the normal and model groups; *, P <0.05 compared with the normal group.

Article Snippet: The diluted primary antibody was added: rabbit antimouse polyclonal antibody SIRT1 (bs-1921R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse monoclonal antibody Bax (ab32503, Abcam Inc., Cambridge, MA, U.S.A.), rabbit antimouse polyclonal antibody Bcl-2 (bs-20351R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse polyclonal antibody p53 (bs-8687R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9485, Abcam Inc., Cambridge, MA, U.S.A.) (1:500) for overnight incubation and a wash with PBS three times at room temperature, each time for 5 min. Rabbit antimouse polyclonal antibody IgG labeled by horseradish peroxidase (HRP) was added and the tissues were shaken and incubated for 1 h at 37°C, washed with PBS three times, with 5 min intervals.

Techniques: Expressing, Immunohistochemistry

Journal: Bioscience Reports

Article Title: Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice

doi: 10.1042/BSR20170695

Figure Lengend Snippet: ( A ) miR-211 expression and mRNA and protein expressions of SIRT1, Bcl-2, Bax, and p53 in mice lens; ( B ) strip chart of SIRT1, Bcl-2, Bax, and p53 proteins; ( C ) expressions of SIRT1, Bcl-2, Bax, and p53 proteins in mice lens; *, P <0.05 compared with the normal group.

Article Snippet: The diluted primary antibody was added: rabbit antimouse polyclonal antibody SIRT1 (bs-1921R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse monoclonal antibody Bax (ab32503, Abcam Inc., Cambridge, MA, U.S.A.), rabbit antimouse polyclonal antibody Bcl-2 (bs-20351R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse polyclonal antibody p53 (bs-8687R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9485, Abcam Inc., Cambridge, MA, U.S.A.) (1:500) for overnight incubation and a wash with PBS three times at room temperature, each time for 5 min. Rabbit antimouse polyclonal antibody IgG labeled by horseradish peroxidase (HRP) was added and the tissues were shaken and incubated for 1 h at 37°C, washed with PBS three times, with 5 min intervals.

Techniques: Expressing, Stripping Membranes

Journal: Bioscience Reports

Article Title: Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice

doi: 10.1042/BSR20170695

Figure Lengend Snippet: ( A ) Sequence of 3′-UTR region of SIRT1 mRNA binding with miR-211 ; ( B ) luciferase activity detection; *, P <0.05 compared with the NC group.

Article Snippet: The diluted primary antibody was added: rabbit antimouse polyclonal antibody SIRT1 (bs-1921R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse monoclonal antibody Bax (ab32503, Abcam Inc., Cambridge, MA, U.S.A.), rabbit antimouse polyclonal antibody Bcl-2 (bs-20351R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500), rabbit antimouse polyclonal antibody p53 (bs-8687R, Beijing Bioss Biotech Co. Ltd., Beijing, China) (1:500) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9485, Abcam Inc., Cambridge, MA, U.S.A.) (1:500) for overnight incubation and a wash with PBS three times at room temperature, each time for 5 min. Rabbit antimouse polyclonal antibody IgG labeled by horseradish peroxidase (HRP) was added and the tissues were shaken and incubated for 1 h at 37°C, washed with PBS three times, with 5 min intervals.

Techniques: Sequencing, Binding Assay, Luciferase, Activity Assay