- PA5-34806 - Invitrogen Antibodies SMAD4 antibody

Submitted references Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

Refaat B, Zekri J, Aslam A, Ahmad J, Baghdadi MA, Meliti A, Idris S, Sultan S, Alardati H, Saimeh HA, Alsaegh A, Alhadrami M, Hamid T, Naeem ME, Elsamany SA
Pathology oncology research : POR 2021;27:1610032

Molecular diversity of diencephalic astrocytes reveals adult astrogenesis regulated by Smad4.

Ohlig S, Clavreul S, Thorwirth M, Simon-Ebert T, Bocchi R, Ulbricht S, Kannayian N, Rossner M, Sirko S, Smialowski P, Fischer-Sternjak J, Götz M
The EMBO journal 2021 Nov 2;40(21):e107532

Obesity reduces mammary epithelial cell TGFβ1 activity through macrophage-mediated extracellular matrix remodeling.

Chamberlin T, Thompson V, Hillers-Ziemer LE, Walton BN, Arendt LM
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020 Jun;34(6):8611-8624

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of SMAD4 using 50 µg of mouse muscle lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a SMAD4 polyclonal antibody (Product # PA5-34806) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of SMAD4 using 30 µg PC-12 whole cell extract. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a SMAD4 polyclonal antibody (Product # PA5-34806) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of SMAD4 using Various whole cell extracts (30 µg). Samples were loaded onto a 10% SDS-PAGE gel and probed with a SMAD4 polyclonal antibody (Product # PA5-34806) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of SMAD4 using 30 µg of A) HeLa and B) HepG2 lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a SMAD4 polyclonal antibody (Product # PA5-34806) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of SMAD4 in Various whole cell extracts (30 µg). Samples were separated by 7.5% SDS-PAGE and the membrane was probed with SMAD4 Polyclonal antibody (Product # PA5-34806) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using SMAD4 Polyclonal Antibody (Product # PA5-34806). Various whole cell extracts (30 µg) were separated by 10% SDS-PAGE, and the membrane was blotted with SMAD4 Polyclonal Antibody (Product # PA5-34806) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HT-29 (Lane 1), HeLa (Lane 2), C2C12 (Lane 3), HEK 293 (Lane 4), MCF7 (Lane 5), U-87 MG (Lane 6), A-431 (Lane 7) and U-2 OS (Lane 8). The blot was probed with Anti-SMAD4 Polyclonal Antibody (Product # PA5-34806, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 60 kDa band corresponding to SMAD4 was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of SMAD4 was performed by separating 30 µg of non-transfected (–) and transfected (+) 293T whole cell extracts by 10% SDS-PAGE. Proteins were transferred to a membrane and probed with a SMAD4 Polyclonal Antibody (Product # PA5-34806) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using SMAD4 Polyclonal Antibody (Product # PA5-34806). Various whole cell extracts (30 µg) were separated by 10% SDS-PAGE, and the membrane was blotted with SMAD4 Polyclonal Antibody (Product # PA5-34806) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of SMAD4 was performed by separating various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a SMAD4 Polyclonal Antibody (Product # PA5-34806) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of SMAD4 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1106592_LV and CRISPR1106595_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of SMAD4 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa SMAD4 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-SMAD4 Polyclonal Antibody (Product # PA5-34806, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to SMAD4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SMAD4 was performed using 70 % confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with SMAD4 Polyclonal Antibody (PA5-34806) at 5 µg/mL in 0.1% BSA and incubated overnight at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear staining. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of SMAD4 was performed in SK-N-SH cells fixed in 4% paraformaldehyde at RT for 15 min. Green: SMAD4 Polyclonal Antibody (Product # PA5-34806) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of SMAD4 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: SMAD4 Polyclonal Antibody (Product # PA5-34806) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Paraffin) analysis of SMAD4 was performed in paraffin-embedded rat kidney tissue using SMAD4 Polyclonal Antibody (Product # PA5-34806) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Paraffin) analysis of SMAD4 was performed in paraffin-embedded mouse testis tissue using SMAD4 Polyclonal Antibody (Product # PA5-34806) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Paraffin) analysis of SMAD4 was performed in paraffin-embedded rat brain tissue using SMAD4 Polyclonal Antibody (Product # PA5-34806) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of paraffin-embedded human ulcerative colitis tissue using SMAD4 Polyclonal Antibody (Product # PA5-34806).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: SMAD4 Polyclonal Antibody detects SMAD4 protein at cytoplasm and nucleus in mouse brain by immunohistochemical analysis. Sample: Paraffin-embedded mouse brain. SMAD4 Polyclonal Antibody (Product # PA5-34806) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: SMAD4 Polyclonal Antibody detects SMAD4 protein at cytoplasm and nucleus in mouse duodenum by immunohistochemical analysis. Sample: Paraffin-embedded mouse duodenum. SMAD4 Polyclonal Antibody (Product # PA5-34806) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 9 Smad4 regulates diencephalic astrocyte proliferation and neurosphere formation A Reactome pathway analysis for all 174 transcription factor genes with higher expression in DIE (upper) and all 205 genes higher in CTX GM (lower) astrocytes. B t-SNE visualization of DIE astrocytes isolated by ACSA-2 MACS (Fig 1A ) showing Smad4 expression (scaled log-normalized read counts). C Example of a Smad4/GFP double-positive astrocyte (indicated by arrowhead) in a single optical section of DIE from 3-mo-old GLAST CreERT2 /eGFP mice 21 dpt immunostained as indicated on top of the panel. Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel. Scale bars: 15 um (left panel) and 10 um (right panel). D Example of a proliferating (PCNA + , arrowhead) astrocyte in a single optical section of DIE from 3-mo-old GLAST CreERT2 /eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels. Scale bars: 15 um. E Histogram depicting percentage of PCNA-positive cells of all GFP-positive cells in the DIE of the genotype indicated on the x-axis ( n = 6 animals per genotype; 5 ROI covering the diencephalon on a total of 3 slides were analysed per animal; PCNA-negative and PCNA-positive cells comparing wt/wt vs. fl/fl animals, adjusted P -value = 0.002). F, G Histograms depicting percentage of the neurosphere-generating cells obtained from the DIE (F) or SEZ (G) of GLAST CreERT2 /Smad4 fl/fl /eGFP

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 1 Relative expression (median) of Smad4 gene and protein in addition to the protein localisation by IHC in the FFPE ( n = 90 patients) and fresh ( n = 40 patients) cohorts of paired malignant and normal colonic tissues according to clinical stages and tumour sidedness (Scale bar = 15 mum; a = p < 0.05 compared with right-sided normal tissue from early cancer; b = p < 0.05 compared with early stage right-sided colon cancer; c = p < 0.05 compared with left-sided normal tissue from early cancer; d = p < 0.05 compared with early stage left-sided colon cancer; e = p < 0.05 compared with right-sided normal tissue from advanced cancer; f = p < 0.05 compared with advanced stage right-sided colon cancer and g = p < 0.05 compared with left-sided normal tissue from advanced cancer).

PA5-34806

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