Immunostaining of phosphorylated Histone H3 (pH3) and pSmad2/3 was performed as previously described with a minor modification [10 (link)]. For pSmad2/3 staining, tadpoles were treated with a permeabilization solution (1% NP-40, 1X PBST) for 30 min after bleaching. The following primary and secondary antibodies were used: anti-pH3 antibody at 1:500 dilution (Upstate Biotechnology); anti-pSmad2/3 antibody at 1:500 dilution (Cell Signaling Technology); Alexa Fluor 488 goat anti-rabbit antibody at 1:500 dilution (Molecular Probe); Alexa Fluor 488 goat anti-mouse antibody at 1:500 dilution (Molecular Probe). Whole-mount in situ hybridization (WISH) and probe synthesis were carried out as described previously [12 (link), 13 (link)]. The tgfb1 antisense and sense probes were generated from pDH105-tgfb1.
Anti psmad2 3 antibody
Manufactured by Cell Signaling Technology
The Anti-pSmad2/3 antibody is a laboratory reagent used to detect and quantify the phosphorylated forms of the Smad2 and Smad3 proteins. Smad2 and Smad3 are key mediators of the transforming growth factor-beta (TGF-β) signaling pathway, and their phosphorylation is a critical step in this pathway. The antibody can be used in various immunoassay techniques, such as Western blotting and immunohistochemistry, to analyze the activation of the TGF-β signaling cascade.
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Lab products found in correlation
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Sp6 rna polymerase, by Roche (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti smad2 3 antibody, by Cell Signaling Technology (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Bca assay kit, by Vazyme (1 mentions)
Internal control gapdh antibody, by Cell Signaling Technology (1 mentions)
4 protocols using anti psmad2 3 antibody
1
Immunostaining and WISH for Histone H3 and pSmad2/3
2
Phosphorylated Smad2 Staining in Embryos
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Embryos and explants were stained for phosphorylated Smad2 as described in van Boxtel et al., 2015 (link). Briefly, samples were fixed overnight in 4% paraformaldehyde (PFA), rinsed in phosphate-buffered saline (PBS) + 0.1% Tween-20 (PBT), and dehydrated to 100% methanol. Prior to staining, samples were rehydrated into PBS, rinsed in PBS + 1% Triton X-100, and incubated in ice-cold acetone at −20°C for 20 min. Samples were then blocked in PBS+ 10% FBS and 1% Triton X-100, and then incubated overnight at 4°C with an anti-pSmad2/3 antibody (Cell Signaling #8828) at 1:1000 in block. Samples were rinsed in PBT/1% Triton X-100 and incubated with Alexa Fluor 488 anti-Rabbit IgG (Invitrogen) at 1:1000. Embryos were co-stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and rinsed in PBS + 1% Triton X-100 prior to mounting in 2% methylcellulose for confocal imaging.
3
Whole-mount in situ Hybridization and Immunostaining of Tadpoles
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Whole-mount in situ hybridization and cryosectioning of hybridized tadpoles were performed as previously described42 (link)–44 (link). Tadpoles were fixed in MEMFA solution for 2 h at room temperature and stored in 100% ethanol at − 20 °C. For anti-sense and sense probes, pDH105-inhba was linearized by BamHI and XbaI and subjected to in vitro transcription by T3 and SP6 RNA polymerases (Roche), respectively. For the whole-mount immunostaining, tadpoles were fixed in MEMFA solution for 30 min at room temperature and stored in 100% methanol at − 20 °C. Whole-mount immunostaining was carried out following our standard protocol for pH3 staining15 (link) and with a minor modification for pSmad2/3 staining17 (link). The following antibodies were used at 1:500 dilution: anti-pH3 antibody (Upstate Biotechnology), anti-pSmad2/3 antibody (Cell Signaling Technology), Alexa Fluor 488 goat anti-rabbit antibody (Molecular Probes, Thermo Fisher Scientific), and Alexa Fluor 488 goat anti-mouse antibody (Molecular Probes, Thermo Fisher Scientific).
4
Immunoblotting Analysis of LTBP-4 and Collagen I
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The cell lysates extracted from the cultured cells were used for immunoblotting analysis. Proteins were extracted from cells, and protein concentration was determined with a BCA Assay Kit (Vazyme, Nanjing, China). Equal amounts of protein were loaded onto 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% bovine serum albumin at room temperature for 1 h. Then, the membranes were incubated overnight at 4 °C with anti-LTBP-4 antibody (1:100; Santa Cruz Biotechnology), goat anti-Collagen type I antibody (1:500; Millipore), anti-SMAD2/3 antibody (1:1000; Cell Signaling Technology), anti-pSMAD2/3 antibody (1:1000; Cell Signaling Technology), and internal control GAPDH antibody (Cell Signaling Technology). After three washes with TBST for 30 min, the PVDF membranes were incubated with appropriate secondary antibodies for 1 h. Detection was performed using an enhanced chemiluminescence system, and the intensity of bands was quantified using the Image-QuantTL software (General Electric Company, CT, USA).
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