Non-treated and Calyculin A treated HEK-293 cells were subjected to SDS PAGE followed by western blot with 80427-2-RR (Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours. The membrane was stripped and re-blotted with Alpha Tubulin (66031-1-Ig) antibody as a loading control.
Non-treated and Calyculin A treated HEK-293 cells were subjected to SDS PAGE followed by western blot with 80427-2-RR (Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours. The membrane was stripped and re-blotted with Alpha Tubulin (66031-1-Ig) antibody as a loading control.
IF Staining of HEK-293 using 80427-2-RR
Immunofluorescent analysis of (4% PFA) fixed TGF beta 1 treated and untreated HEK-293 cells using Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody (80427-2-RR, Clone: 240826D11 ) at dilution of 1:500 and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2).
Immunofluorescent analysis of (4% PFA) fixed TGF beta 1 treated and untreated HEK-293 cells using Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody (80427-2-RR, Clone: 240826D11 ) at dilution of 1:500 and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2).
FC experiment of HEK-293 using 80427-2-RR
1X10^6 HEK-293 cells untreated (dashed lines) or treated with Calyculin A were intracellularly stained with 0.13 ug Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Recombinant antibody (80427-2-RR, Clone:240826D11) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2)(red), or 0.13 ug Rabbit IgG Isotype Control Recombinant Antibody (98136-1-RR, Clone: 240953C9) (blue). Cells were fixed with 4% PFA and permeabilized with 90% MeOH.
1X10^6 HEK-293 cells untreated (dashed lines) or treated with Calyculin A were intracellularly stained with 0.13 ug Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Recombinant antibody (80427-2-RR, Clone:240826D11) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2)(red), or 0.13 ug Rabbit IgG Isotype Control Recombinant Antibody (98136-1-RR, Clone: 240953C9) (blue). Cells were fixed with 4% PFA and permeabilized with 90% MeOH.
ChIP experiment of HaCaT using 80427-2-RR
Chromatin was prepared from HaCaT cells treated with TGF-β1 (7 ng/ml) for 1 h. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 20 µg of cross-linked chromatin, 5 µg of Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (80427-2-RR) or 5 ug of Normal Rabbit IgG (98136-1-RR), and 20 µl of Protein A Magarose Beads. The immunoprecipitated DNA was quantified by real-time PCR.
Chromatin was prepared from HaCaT cells treated with TGF-β1 (7 ng/ml) for 1 h. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 20 µg of cross-linked chromatin, 5 µg of Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (80427-2-RR) or 5 ug of Normal Rabbit IgG (98136-1-RR), and 20 µl of Protein A Magarose Beads. The immunoprecipitated DNA was quantified by real-time PCR.
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80427-2-RR targets Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) in WB, IF/ICC, FC (Intra), ELISA, ChIP-qPCR applications and shows reactivity with human samples.
PBS with 0.02% sodium azide and 50% glycerol, pH 7.3.
Storage Conditions
Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20oC storage. 20ul sizes contain 0.1% BSA.
Background Information
SMAD2, also named as MADH2 and MADR2, belongs to the dwarfin/SMAD family, contains 1 MH1 (MAD homology 1) domain and 1 MH2 (MAD homology 2) domain. SMAD2 is a receptor-regulated SMAD(R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta and activin type 1 receptor kinases. This protein may act as a tumor suppressor in colorectal carcinoma. It is phosphorylated on one or several of Thr-220, Ser-245, Ser-250, and Ser-255. In response to TGF-beta, It is phosphorylated on Ser-465/467 by TGF-beta and activin type 1 receptor kinases, and then able to interact with SMURF2, recruiting other proteins, such as SNON, for degradation. In response to decorin, the naturally occurring inhibitor of TGF-beta signaling, it is phosphorylated on Ser-240 by CaMK2. It is phosphorylated by MAPK3 upon EGF stimulation; which increases transcriptional activity and stability, and is blocked by calmodulin. In response to TGF-beta, it is ubiquitinated by NEDD4L, which promotes its degradation. In response to TGF-beta signaling, it is acetylated on Lys-19 by coactivators, which increases transcriptional activity. The molecular weight of unphosphorylated forms of Smad2 is 52 kDa and phosphorylated forms of Smad2 is 58 kDa. (PMID: 9006934)
Protocols
Product Specific Protocols
WB protocol for Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody 80427-2-RR
Non-treated and Calyculin A treated HEK-293 cells were subjected to SDS PAGE followed by western blot with 80427-2-RR (Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours. The membrane was stripped and re-blotted with Alpha Tubulin (66031-1-Ig) antibody as a loading control.
IF/ICC Figures
IF Staining of HEK-293 using 80427-2-RR
Immunofluorescent analysis of (4% PFA) fixed TGF beta 1 treated and untreated HEK-293 cells using Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) antibody (80427-2-RR, Clone: 240826D11 ) at dilution of 1:500 and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2).
FC (INTRA) Figures
FC experiment of HEK-293 using 80427-2-RR
1X10^6 HEK-293 cells untreated (dashed lines) or treated with Calyculin A were intracellularly stained with 0.13 ug Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Recombinant antibody (80427-2-RR, Clone:240826D11) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2)(red), or 0.13 ug Rabbit IgG Isotype Control Recombinant Antibody (98136-1-RR, Clone: 240953C9) (blue). Cells were fixed with 4% PFA and permeabilized with 90% MeOH.
CHIP-QPCR Figures
ChIP experiment of HaCaT using 80427-2-RR
Chromatin was prepared from HaCaT cells treated with TGF-β1 (7 ng/ml) for 1 h. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 20 µg of cross-linked chromatin, 5 µg of Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (80427-2-RR) or 5 ug of Normal Rabbit IgG (98136-1-RR), and 20 µl of Protein A Magarose Beads. The immunoprecipitated DNA was quantified by real-time PCR.
The species listed in Tested Reactivity are in-house verified and applicable species. For unlisted species, please refer to the homology analysis of the immunogen sequence and related species. For rabbit polyclonal antibodies, homology >70% is recommended. For mouse monoclonal antibodies and rabbit recombinant antibodies, homology >90% is recommended. Generally, the higher the homology, the greater the applicability. However, there will be certain differences in protein expression in different species, tissues or cells. Therefore, the homology analysis results are for reference only and do not serve as a guarantee.
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