P smad

Manufactured by Cell Signaling Technology

Sourced in United States

P-Smad is a lab equipment product that detects and quantifies phosphorylated Smad proteins. Smad proteins are key mediators of the TGF-beta signaling pathway, which regulates various cellular processes. The P-Smad product can be used to measure the activation state of Smad proteins in biological samples.

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Lab products found in correlation

β actin, by Santa Cruz Biotechnology (2 mentions) P akt, by Cell Signaling Technology (2 mentions) N cadherin, by Cell Signaling Technology (2 mentions) Vimentin, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Sostdc1, by Abcam (1 mentions) Lamin b, by Cell Signaling Technology (1 mentions) Dnmt1, by Abcam (1 mentions) Ecl substrate, by Merck Group (1 mentions) Ripa lysis buffer, by Solarbio (1 mentions) Non phospho β catenin, by Cell Signaling Technology (1 mentions) Western blocking solution, by Roche (1 mentions) Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Total p38, by Cell Signaling Technology (1 mentions) Total jnk, by Cell Signaling Technology (1 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions) P erk, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

P-Smad) 1/5/8 antibody

16 protocols using p smad

Western Blot and ELISA Analysis

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Harvested cells and frozen tissues were treated with RIPA lysis buffer (Solarbio, China) for protein extraction. Protein lysate was loaded for SDS-PAGE, and was transferred from the gel to the membrane. Unspecific binding was blocked with 5% skimmed milk. Proteins were incubated with β-actin (Santa Cruz), tubulin, FPN (Sigma), DNMT1, SOSTDC1 (Abcam), Lamin B, p-SMAD, SMAD, G9a (Cell signaling), and E4BP4 (Abcam) antibodies at 4 °C overnight. Membranes were then incubated for 1 h with secondary antibodies. Protein detection was performed with the ECL substrate (Millipore, USA), and β-actin was used as the internal control. ELISA kits (R&D Systems) were used to detect levels of BMP4, BMP7, IL-6, and hepcidin.

Zhou Q., Chen J., Feng J, & Wang J. (2018). E4BP4 promotes thyroid cancer proliferation by modulating iron homeostasis through repression of hepcidin. Cell Death & Disease, 9(10), 987.

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Immunohistochemical Analysis of Dental Samples

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Immunostaining and routine histological analyses by H and E staining were carried out, as previously described [51 (link)]. Calcified teeth were sectioned following decalcification using 0.5 M EDTA for three weeks. For immunostaining, sections were first rehydrated and then processed for antigen retrieval. Blocking was performed by incubating the sections in the 1X western blocking solution (Roche, Mannheim, Germany; Ref. 11921673001) for one hour at room temperature. The primary antibodies used in this study were directed against Ki67 (Thermo Scientific, Waltham, MA, USA, cat. no. RM-9106-s), non-phospho β-catenin (Cell Signaling Technology, Danvers, MA, USA, cat. no. 8814S), and pSMAD (Cell Signaling Technology, Danvers, MA, USA; cat. no. 9511S). The secondary antibodies used in this study were biotinylated goat anti-rabbit IgG (Invitrogen, Waltham, MA, USA) and goat anti-rabbit IgG Flamma 488 (BioActs, Incheon, Korea, cat. no. RSA1241).

Neupane S., Aryal Y.P., Kim T.Y., Yeon C.Y., An C.H., Kim J.Y., Yamamoto H., Lee Y., Sohn W.J, & Kim J.Y. (2020). Signaling Modulations of miR-206-3p in Tooth Morphogenesis. International Journal of Molecular Sciences, 21(15), 5251.

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Western Blot Analysis of Inflammatory Signaling

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Expression of activated pro-inflammatory (p38 and JNK) and anti-inflammatory proteins (ERK, SMAD) was determined using our standard Western blot technique (17 (link)). In brief, arterial tissue was collected, preserved in RNAlater, and stored at −80^oC until homogenization. Post homogenization, proteins were estimated using Bradford Assay. Proteins were then analyzed using 10% SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane (Bio-Rad) using a Bio-Rad Semi-Dry Transfer Cell at 15V for 45 minutes. Membranes were blocked for 1 hour at room temperature with 5% milk, decanted, and incubated overnight with the appropriate primary antibody in 5% milk: p- JNK (Cell Signaling, 1:1000, #4668S), Total JNK (Cell Signaling, 1:1000, #9252S), p- p38 (Cell Signaling, 1:1000, #4631S), Total p38 (Cell Signaling, 1:1000, #9212S), p- ERK (Santa Cruz, 1:1000, sc7383), β-actin (1:1000, Cell Signaling, #4967L) or p-SMAD (Cell Signaling, 1:1000, #13820S). After overnight incubation, membranes were washed and incubated with HRP-conjugated anti-rabbit IgG (Cell Signaling, 1:1000, #7074S) or anti-mouse IgG (Santa Cruz, 1:1000, sc2064) for one hour at room temperature. Membranes were washed and incubated with ECL (Thermo Scientific, #32106). Densitometry analysis of protein expression was completed using Image J as previously reported (9 (link)).

Shoulders H., Garner K.H, & Singla D.K. (2018). Macrophage Depletion by Clondronate Attenuates BMP-7 Induced M2 Macrophage Differentiation and Improved Systolic Blood Velocity in Atherosclerosis. Translational research : the journal of laboratory and clinical medicine, 203, 1-14.

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Immunoblot Analysis of Cell Signaling

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Cell lysates were prepared at 75% of confluence by using 500 μL of radio-immunoprecipitation assay buffer (25 mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) in 10-cm culture dishes with a 20-minute incubation on ice. The protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Hercules, CA). Immunoblot analyses were performed as previously described [37 (link), 38 (link)]. β-Actin antibodies were obtained from Santa Cruz Biotechnology. HER2, Claudin-1, ZEB1, ZO-1, Fak, pFak, Smad, pSmad were purchased from Cell Signaling Technology. The secondary antibodies were the F(ab)₂ fragment of donkey anti-mouse immunoglobulin (product NA931) or of donkey anti-rabbit immunoglobulin (product NA9340) linked to horseradish peroxidase from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The immunoblot reagents were from an electrochemiluminescence kit (Amersham Biosciences, NJ, USA).

Hou J., Zhou Z., Chen X., Zhao R., Yang Z., Wei N., Ni Q., Feng Y., Yu X., Ma J, & Guo X. (2016). HER2 reduces breast cancer radiosensitivity by activating focal adhesion kinase in vitro and in vivo. Oncotarget, 7(29), 45186-45198.

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Colorectal Cancer Cell Line Characterization

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Human colorectal adenocarcinoma cell lines HCT116 and SW480 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Antibodies against PARP-1 (sc-8007), PCNA (sc-56), α-tubulin (sc-5286), ZO-1 (sc-33725), p-GSK3β (sc-135653), and Twist (sc-15393) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against claudin (#132552), p-Akt (#9271), Slug (#9585), Snail (#3879), N-cadherin (#13116), E-cadherin (#3196), and p-Smad (#9511) were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant human TGF beta 1 protein (TGF-β) was purchased from Abcam (Cambridge, UK).

Chei S., Oh H.J., Song J.H., Seo Y.J., Lee K, & Lee B.Y. (2019). Magnolol Suppresses TGF-β-Induced Epithelial-to-Mesenchymal Transition in Human Colorectal Cancer Cells. Frontiers in Oncology, 9, 752.

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Signaling Pathways in BMP2-Induced Osteogenesis

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Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline, penicillin–streptomycin, and 0.25% trypsin-EDTA were obtained from GIBCO-BRL (Grand Island, NY). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Seoul, Korea). Emerald Amp GRPCR Master Mix was purchased from TaKaRa (Shiga, Japan), and AmpiGene^TM qPCR Green Mix Hi-ROX was purchased from Enzo (Farmingdale, NY). Recombinant human BMP2 was purchased from Cowellmedi Co. (Busan, Korea). Antibodies against Prx II and β-actin were obtained from Santa Cruz Biotechnology (Dallas, TX). Antibodies against PP2A Cα, Smad, and phospho-Smad (p-Smad) were purchased from Cell Signaling Technology (Cambridge, MA).

Kim K.M., Kim D.Y., Lee D.S., Kim J.W., Koh J.T., Kim E.J, & Jang W.G. (2019). Peroxiredoxin II negatively regulates BMP2-induced osteoblast differentiation and bone formation via PP2A Cα-mediated Smad1/5/9 dephosphorylation. Experimental & Molecular Medicine, 51(6), 62.

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Recombinant BMP-2 Signaling Pathway

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Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) was purchased from R&D Systems (Minneapolis, MN, USA). Penicillin, streptomycin, cell culture medium, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies against the following proteins were purchased from the indicated companies: actin, Smad, and secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA); p‐Smad, p‐AKT, AKT, MAP kinases, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; Cell Signaling Technology, Beverly, MA, USA).

Kim S., Yuk H.J., Ryu H.W., Oh S., Song D.Y., Lee K., Park K., Choi S, & Seo W.D. (2019). Biofunctional soyasaponin Bb in peanut (Arachis hypogaea L.) sprouts enhances bone morphogenetic protein‐2‐dependent osteogenic differentiation via activation of runt‐related transcription factor 2 in C2C12 cells. Phytotherapy Research, 33(5), 1490-1500.

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Protein Isolation and Western Blotting

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Soluble cytoplasmic proteins were isolated from DFCs with a specific protein isolation buffer (20 mM Tris/HCl pH 8.0, 137 mM NaCl, 48 mM NaF, 2 mM Na₃VO₄, 1% NP-40, 10% glycerol, 1% phosphatase inhibitor cocktail 3 from Sigma, 1 cOmplete™ mini protease inhibitor cocktail tablet per 10 ml from Sigma). Samples were separated by SDS–polyacrylamide gel electrophoresis in 4–15% Mini PROTEAN^® TGX Stain-Free™ Protein Gels (BIO-RAD) which were then activated with UV light before blotting the proteins to a nitrocellulose membrane as described before (Morsczeck et al. 2019b (link)). Images of total lane protein were taken. Primary antibodies for SMAD 1 (Cell Signaling #6944) and SMAD 1/5 phosphorylated at Ser463/465 (pSMAD, Cell Signaling #9516) and HRP-linked secondary antibodies were used according to the manufacturers’ instructions (Cell Signaling). The detection of antibody-marked protein bands was performed by chemiluminescence signaling developed with Clarity Western ECL Substrate (BIO-RAD) and measured with the ChemiDoc Touch Imaging System (BIO-RAD). Total lane protein was used as the loading control.

Pieles O., Reck A, & Morsczeck C. (2020). High endogenous expression of parathyroid hormone-related protein (PTHrP) supports osteogenic differentiation in human dental follicle cells. Histochemistry and Cell Biology, 154(4), 397-403.

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Western Blot Analysis of Signaling Pathways

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Expression of total Smad and phospho-Smad (p-Smad), total P38 and phosphor-P38 (p-P38) and BMP4, and UCP-1 was measured by Western blotting. Protein was separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Antibodies of BMP4, Smad, p-Smad, P38, and p-P38 were purchased from Cell Signaling Technology (USA). The antibody of UCP-1 was purchased from ABclonal (USA). Antibodies of α-tubulin and GAPDH were purchased from Proteintech (USA). BMP4, P38, p-P38, p-Smad, Smad, and UCP-1 expression were analyzed using a rabbit-antibody (1 : 1000) primary antibody, and a rabbit-anti mouse IgG-HRP (1 : 1000) secondary antibody. GAPDH and α-tubulin (1 : 1000 dilution for the primary antibody and 1 : 1000 dilution for the secondary antibody) were used as controls. Western blot bands were scanned and quantified by an image analyzer, Quantity One System (Odyssey, USA).

Wang X., Ma B., Chen J., You H., Sheng C., Yang P, & Qu S. (2021). Glucagon-like Peptide-1 Improves Fatty Liver and Enhances Thermogenesis in Brown Adipose Tissue via Inhibiting BMP4-Related Signaling Pathway in High-Fat-Diet-Induced Obese Mice. International Journal of Endocrinology, 2021, 6620289.

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Analyzing JAK/STAT and TGF-β Pathways in Hut78 Cells

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The human TCL cell line Hut78 was obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal mono-nuclear cells were separated from the peripheral blood of healthy volunteers. The anti-mPGES-1 antibody (#10004350) and mPGES-1 inhibitor CAY10526 (#10010088) were ordered from Cayman Chemical Company (Ann Arbor, MI, USA). Antibodies to JAK2 (#3230), STAT3 (#9139), p-STAT3 (#52075), Akt (#4691), p-Akt (#4060), TGF-β (#3709), Smad (#9523) and p-Smad (#9520) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies to JAK1 (ab133666), GAPDH (ab181602) and Caspase-3 (ab32351) were purchased from Abcam (Cambridge, MA, USA).

Li Y., Qiu M., Li B., Xiao J., Yang W., Xie S., Du Y., Huang K, & Nie D. (2022). Growth of T-cell lymphoma cells is inhibited by mPGES-1/PGE2 suppression via JAK/STAT, TGF-β/Smad3 and PI3K/AKT signal pathways. Translational Cancer Research, 11(7), 2175-2184.

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