44-244G

antibody from Invitrogen Antibodies

Targeting: SMAD2 JV18-1, MADH2, MADR2

Provider product page for 44-244G

Western blot

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [27]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Immunohistochemistry [2]
Flow cytometry [1]
Chromatin Immunoprecipitation [1]
Other assay [20]

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Product number: 44-244G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-SMAD2 (Ser465, Ser467) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

SMAD2 protein structure - 44-244G shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of SMAD2 (pSpS465/467)showing staining in the nucleus of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a SMAD2 (pSpS465/467) polyclonal antibody (Product # 44-244G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of SMAD2 (pSpS465/467) showing staining in the nucleus of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a SMAD2 (pSpS465/467) polyclonal antibody (Product # 44-244G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Effects of anti-Nodal 3D1 antibody on cell signaling and cell cycle related molecules A. Levels of P-Smad2 and P-ERK1/2 are reduced within 4hr of 3D1 mAb treatment (4 mug/ml) in C8161 human melanoma cells compared to IgG treated control. B. After 72 hr of 3D1 mAb treatment there is a reduction of Nodal, Cyclin B1 and P-H3 with a concomitant increase in p27 in C8161 cells compared to IgG treated control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 In vivo effects of anti-Nodal 3D1 mAb on C8161 human melanoma cells A. Significantly reduced tumor volumes are observed in C8161 Nude mice orthotopic xenografts treated with direct tumor injections of 3D1 mAb versus control IgG. Histograms represent mean values +/- SD. Representative IHC staining in B. shows reduced nuclear expression (activation) of Smad2 in C8161 orthotopic xenograft Nude mouse treated with 3D1 mAb compared to IgG control (20X original magnification). The potential for lung colonization (shown microscopically with H&E staining) of C8161 cells (after systemic introduction) in Nude mice C. (40X original magnification) is significantly reduced in animals treated with IP administration of 3D1 mAb vs IgG control D. Histograms represent mean values +/- SEM. Representative IHC staining in E. shows reduced nuclear expression (activation) of P-Smad2 in C8161 lung colony of a 3D1 mAb treated Nude mouse versus IgG control (63X original magnification). (* P < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 In vivo effects of anti-Nodal 3D1 mAb on A375SM human melanoma cells A. After 8 days, mean tumor volume of A375SM orthotopic Nude mice xenografts was significantly smaller in 3D1 mAb treated than in IgG control treated animals. Tumor volumes in dabrafenib (BRAFi) treated animals also showed a trend towards reduced tumor volumes compared to control. Histograms represent mean values +/- SD. B. Representative IHC of P-Smad2 showing nuclear staining in A375SM orthotopic xenografts in Nude mice treated with control IgG, 3D1 mAb or BRAFi (* P < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Co-culture (CoC) experiments. ( A ) Schematic diagram summarizing the co-culture experiments using HeMn melanocytes and C8161 melanoma cells. Lysates of cells growing on the bottom well (red square) were used for Western blotting; ( B ) Western blot results show increased expression of P-SMAD2 and P-ERK1/2 in HeMn melanocytes co-cultured with human C8161 melanoma cells compared to control HeMn cells culured alone. However, Nodal expression was not detected in HeMn cells grown alone or in co-culture with C8161 melanoma cells; and ( C ) Results from WB analysis show reduced levels of Nodal, P-SMAD2 and P-ERK1/2 in C8161 melanoma cells when co-cultured with HeMn melanocytes. Numbers above WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Effect of treatment of C8161 melanoma cells with HeMn melanocyte conditioned medium (HeMn-CM). ( A ) C8161 cells treated with HeMn-CM show morphologic changes such as dendritic shape with long cytoplasmic extensions reminiscent of melanocyte differentiation. (40x original magnification); ( B ) Although, Melanoma Differentiation Associated protein 7 (MDA-7) was detected in C8161 cells treated with HeMn-CM, the melanin synthesis associated enzyme tyrosinase was not; ( C ) C8161 cells treated with HeMn-CM showed similar proliferation rate and cell viability as non-treated control C8161 cells making potential toxic effect of HeMn-CM on C8161 unlikely; ( D ) Western blot analysis show that HeMn-CM treated C8161 express lower levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated control C8161 cells. Numbers below WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 HeMn melanocyte derived melanosomes (MDM) affect Nodal expression and signaling components in C8161. ( A ) Microphotograph (40x original magnification) shows MDM containing globules (red arrows) produced by HeMn melanocytes; ( B ) C8161 melanoma cells treated with different concentrations of MDM collected from HeMn melanocytes showed reduction in Nodal expression compared to untreated control C8161 cells. To mimic the anti-oxidant effect of free radical scavengers contained in MDM, C8161 cells were treated with N -acetyl- l -cysteine (NAC) known to generate the anti-oxidant, glutathione; ( C ) Western Blot analysis show that treatment of C8161 melanoma cells with 5 mM NAC resulted in reduced levels of Nodal, P-SMAD and P-ERK1/2 compared to untreated control C8161 cells; ( D ) Treatment of C8161 cells with synthetic melanin, also known to have anti-oxidant properties, resulted in a dose-dependent reduction of Nodal, P-SMAD and P-ERK1/2 levels compared to untreated control C8161 cells. Numbers above WB bands represent densitometric units, normalized to actin loading control, total SMAD2/3 or total ERK1/2, as appropriate, relative to control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Pbsn-cre4Prb/+;PI3K GOF/+ mouse prostatic stroma has increased levels of p-SMAD2, a biomarker of TGF-beta activity. (A) Dorsal prostate sections from 4-, 7- and 12-month-old Pbsn-cre4Prb+/+;PI3K GOF/+ (control) and Pbsn-cre4Prb/+;PI3K GOF/+ male mice were stained with an antibody recognizing the phosphorylated form of SMAD2 (p-SMAD2) and DAPI to mark nuclei. Slides were imaged using fluorescence microscopy. (B) The frequency of p-SMAD2-positive stromal cells began to differ between groups starting at 7 months and continuing until at least 12 months of age. (C) Pbsn-cre4Prb/+;PI3K GOF/+ male mice were stained using RNAscope(r) to visualize Tgfb1 mRNA. Representative image of greater Tgfb1 mRNA abundance in the epithelium of Pbsn-cre4Prb/+;PI3K GOF/+ mice. Mean +- SEM of two to four mice. Significant differences among groups: n.s., not significant; * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Compound 145 limits TGF-beta 1 -induced Smad-2 phosphorylation and nuclear translocation in MRC-5 cells. Lung fibroblasts were pre-incubated with the studied compounds (10 µM) for 1 h, and then TGF-beta 1 (5 ng/mL) was added for 1 h. ( A ) Representative immunoblot and densitometry analysis of p-Smad-2 in MRC-5 ( n = 4). The relative optical density (ROD) signal was normalized to the GAPDH control level. Each bar represents the mean value (+- S.E.M.). ( B ) Immunofluorescence staining for p-Smad-2 protein: MRC-5 were fixed, permeabilized, blocked, and incubated with anti-p-Smad-2 antibody followed by Alexa Fluor 546 conjugated antibody, nuclei were stained with the Hoechst 33342 dye. Microphotographs were taken using Leica DMi8 microscope with Thunder Imaging System, 40x objective, bar = 50 um. ( C ) Cells exhibiting strong nuclear p-Smad-2 signals were counted in 20 randomly selected fields of view and expressed as a percentage fraction in the entire MRC-5 population. The results were considered statistically significant at the p level of 0.05, against the control (#) and TGF-beta 1 (*) conditions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Smad2 and Smad3 expression in placentas across gestation. Healthy placentas from 6 to 39 weeks of pregnancy were assessed for total and phosphor-(p) Smad2 (A) and Smad3 (B) levels using Western Blotting. Representative blots and linear regression graphs depicting the relationship between their levels and gestational stage. The number on the top of each lane indicates gestational age of the sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Differential cellular localization of the active forms of Smad2 (A) and Smad3 (B) in anchoring villi. Immunofluorescence staining was performed on 10-14 week placentas ( n = 3) using antibodies against pSmad2 or pSmad3, as well as HLA-G to identify EVTs. Nuclei were stained with DAPI. Representative pictures from a 12-week placenta are shown. Nuclear staining for pSmad2 was more prominent in HLA-G negative cells in the proximal column cells (CC, yellow arrow), while pSmad3 (green arrow) was present mostly in nuclei of cells in the distal region where HLA-G positive EVT cells are present (white arrow). Scale bar = 40 mum. STB, syncytiotrophoblast.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Active Smad3 is downregulated in preeclamptic placentas. Placentas from PE patients and gestational age-matched controls were assessed for total and phosphorylated (p) Smad2 (A) and Smad3 (B) by Western Blotting. Total Smad2 and Smad3 levels, after normalizing to their respective GAPDH signal, showed no significant difference between the PE and control placentas. The ratio of pSmad2/total Smad2 was also not significantly different between the PE and the corresponding control. However, pSmad3/total Smad3 was significantly decreased (*** p < 0.001) in PE placentas relative to their gestational age-matched controls ( n = 3). The number on the top of each lane indicates gestational age of the sample. PTB, preterm birth; EOPT, early onset preeclampsia; Term, healthy term placentas; LOPE, late onset preeclampsia.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Effect of sauchinone on AngII-induced mesangial cell fibrosis. ( A ) Western blotting and ( B ) real-time PCR showed protein and mRNA levels of fibronectin, Collagen IV, and CTGF in sauchinone-treated and AngII-stimulated cells at 48 h. ( C , D ) Effect of sauchinone on the relative levels of TGF-beta1/Smads. The results were detected by Western blotting and real-time PCR. ( E ) Immunofluorescent images of p-Smad-2 nuclear translocation under the laser scanning confocal microscopy are show (magnification. 400x). Nuclei were stained with DAPI (blue) and p-smad-2 was stained with Alexa Fluor 488 (green). Green fluorescence indicates localization of p-Smad-2. Respective blot data were obtained from five independent experiments. Each value represents the means +- S.E. of five independent experiments. ** p < 0.01 vs. control and ## p < 0.01, # p < 0.05 vs. AngII alone.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Myeloid-lineage cells and transforming growth factor- beta 1 (TGF- beta 1) activity were increased in the periosteum with mechanical loading. A-F) Mice underwent 3 d or one week of axial compression loading of the tibiae. Non-loaded tibiae were used as controls. A) Immunofluorescent staining and B) quantification of tdTomato + (red) cells on the periosteal tibial surface in LysM-cre::Ai14 mice. Blue indicates 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei. Scale bar, 50 um. C) Tartrate-resistant acid phosphatase (TRAP) (magenta) staining and D) quantification on periosteal tibial surface in WT mice. Scale bar, 50 um. E) Immunohistochemical staining and F) quantification of pSmad2 + cells (brown) on the periosteal tibial surface in WT mice. Scale bar, 50 um. C, cortical bone; P, periosteum; M, muscle. 3d, 3 days; 1w, one week. G) WT mice underwent one week of axial compression loading of the tibiae. Non-loaded tibiae were used as controls. mRNA expression of Tgfb1 by reverse transcription quantitative polymerase chain reaction (RT-qPCR) extracted from tibial cortical bone. The analyses of (A-F) were performed on the cross sections of posterior and lateral surfaces of tibiae at 5 mm proximal to the distal TFJ. Loaded tibiae values were normalized to the corresponding non-loaded tibiae values. Data are presented as mean +- SEM. n = 5 mice at different time points (B) n = 7 at different time points (D, F) and n = 3 mice (G). B,D,F) * p < 0.05 compared with the correspond

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 Snail suppresses GBM in vivo. a Tumor formation in orthotopically xenografted immunodeficient mice. b Immunohistochemistry of GFP, Ki67, Nestin, and phospho-SMAD2. c Immunohistochemistry quantification: (left) % Ki67-positive nuclei expressed as a percentage of tumor area; (center and right) reciprocal intensity of Nestin and phosho-SMAD2. Data show mean +- SD of 9-20 fields of 3 mice. Statistical comparison (one-way Anova) indicates significant differences at: * p < 0.05, ** p < 0.01, *** p < 0.001

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Ductal deletion of Hnf1b leads to enhanced signaling pathways that favor tumorigenesis. ( A ) RT-qPCR of YAP transcriptional targets. ( B-E ) CTGF (brown) immunohistochemistry. A strong ectopic CTGF staining was observed in ( C' ) acinar cells in mutants in ( D' ) the epithelium of metaplastic ducts and ( E' ) in PanIN. ( F ) RT-qPCR of the Notch pathway. ( G and H ) HEY2 immunohistochemistry. ( I ) RT-qPCR of TGF-beta1 . ( J and K ) Phospho-SMAD2 (Ser465, Ser467) immunohistochemistry. ( L ) RT-qPCR of EGFR . ( M and N ) Phospho-AKT (Ser473) immunohistochemistry. Nuclei were counterstained with hematoxylin. Scale bars : 100 mum. Control, n >= 3; mutant, n >= 3 for immunohistochemistry; and control, n = 6; mutant, n = 5 for RT-qPCR. * P < .05; ** P < .01; *** P < .001.