Rabbit anti p smad1 5

Manufactured by Cell Signaling Technology

Rabbit anti-p-SMAD1/5 is a primary antibody that specifically recognizes phosphorylated forms of SMAD1 and SMAD5 proteins. SMAD1 and SMAD5 are key intracellular mediators of the bone morphogenetic protein (BMP) signaling pathway.

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5 protocols using rabbit anti p smad1 5

Western Blot Analysis of Signaling Proteins

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Cell lysate was run on an SDS-PAGE gel in ice and blotted onto Immun-Blot PVDF membrane (Bio-Rad) according to the standard protocol. Primary antibodies used in this study are: rabbit anti-BMB2 (Abcam, #14933), rabbit anti-BMP6 (Abcam, #155963), rabbit anti-SIN3A (Cell Signaling Technology, #8056), rabbit anti-p-SMAD1 (Cell Signaling Technology, #11971), rabbit anti-p-SMAD1/5 (Cell Signaling Technology, #9516), rabbit anti-p-SMAD1/5/9 (Cell Signaling Technology, #11971), mouse anti-SMAD1 (Santa Cruz Biotechnology, #7965), rabbit anti-H3K9ac (Cell Signaling Technology, #9649), rabbit anti-H3K4me1 (Cell Signaling Technology, #5326), rabbit anti-H3K9me2 (Cell Signaling Technology, #9725), rabbit anti-FAM83G (Bethyl Laboratories, #A304-282A), rabbit anti-FOXM1 (Cell Signaling Technology, #5436), rabbit anti-p21 (Abcam, #109520), rabbit anti-p38γ (Cell Signaling Technology, #2307), rabbit anti-phospho-p38 (Cell Signaling Technology, #4511), and rabbit anti-GAPDH (Cell Signaling Technology, #5174).

Min D., Byun J., Lee E.J., Khan A.A., Liu C., Loudig O., Hu W., Zhao Y., Herlyn M., Tycko B., Cole P.A, & Ryu B. (2021). Epigenetic Silencing of BMP6 by the SIN3A-HDAC1/2 Repressor Complex Drives Melanoma Metastasis via FAM83G/PAWS1. Molecular cancer research : MCR, 20(2), 217-230.

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Immunostaining of Drosophila Testes

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Fly testes were prepared and immunostained as previously described (Wang and Mann 2003 (link)). The following antibodies were used: 1B1 (1:50, DSHB), rabbit anti-Bam (1:2000, a gift from Chen Dahua), mouse anti-BrdU (1:200, BD), mouse anti-β-Gal (40-1a 1:100, DSHB), rabbit anti-pSmad1/5 (1:200, Cell Signaling#9516S), rabbit anti-STAT [1:4000; against (KLH)-GMADFDTITNFENF-OH], rabbit anti-Vasa [1:8000; against (KLH)-MSDDWDDEPIVDTRGARC-OH], guinea pig anti-Vasa (1:4000; against 6xHis-Vasa produced in Escherichia coli), rabbit anti-Zfh1 (1:4000; against 648–775 aa produced in E. coli, a gift from Zhao Yun), rabbit anti-Tj [1:1000; against (KLH)-CHHQWHMDERRLQPLSPP-OH and (KLH)-CAVEVSPSPHIKLRSFSG-OH].

Tang Y., Geng Q., Chen D., Zhao S., Liu X, & Wang Z. (2017). Germline Proliferation Is Regulated by Somatic Endocytic Genes via JNK and BMP Signaling in Drosophila. Genetics, 206(1), 189-197.

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Signaling Pathway Protein Expression

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Samples were lysed and analysed by western blotting to determine the expression levels of the indicated proteins. The following antibodies were used. Mouse anti‐c‐MYC (1:1000), mouse anti‐HES1 (1:1000) and mouse anti‐Dishevelled (1:1000) were from Santa Cruz Biotechnology. Rabbit anti‐p‐SMAD1/5 (1:1000), rabbit anti‐β‐catenin (1:1000) and rabbit anti‐GAPDH (1:1000) were from Cell Signalling Technology. Rabbit anti‐LRP6 (1:1000 for western blot and 1:200 for immunostaining) and mouse anti‐LGR5 (1:1000 for western blot and 1:200 for immunostaining) antibodies were purchased from Immunoway. Mouse anti‐clathrin (1:200 for immunostaining) and mouse anti‐caveolin (1:200 for immunostaining) were purchased from Proteintech.

Zhang L., He Y., Dong L., Liu C., Su L., Guo R., Luo Q., Gan B., Cao F., Wang Y., Song H, & Li X. (2023). Perturbation of intestinal stem cell homeostasis and radiation enteritis recovery via dietary titanium dioxide nanoparticles. Cell Proliferation, 56(8), e13427.

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Astrocyte Proliferation and Smad1/5 Signaling

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Aldh1l1-eGFP were perfused with PBS followed by 4% PFA. Brains were postfixed in 4% PFA overnight at 4C, equilibrated in 30% sucrose, mounted in O.C.T. (Tissue-Tek) and sectioned into 10 µm thick sagittal cryosections. Sections were blocked with 10% goat serum in 0.1% PBST then stained with 1∶1000 chicken-anti-GFP (EMD Millipore AB16901), 1∶1500 rabbit-anti-Psmad1/5 (Cell Signaling 9516), 1∶300 rabbit-anti-ki67 (Cell Signaling 12075S) and 1∶2000 mouse-anti-GFAP (Sigma G3893) antibodies overnight at 4C. Alexa594, 568, 647 or 488 conjugated secondary antibodies were then added at 1∶1000 for 2 hrs at room temperature and sections were mounted in DAPI Fluoromount-G (Southern Biotech). Image acquisition was done at 20x on a Zeiss Axiocam microscope and expression quantified using ImageJ. Simultaneous staining with rabbit-anti-p-smad1/5 and rabbit-anti-ki67 was done using a sequential technique. P-smad1/5 staining and visualization with an Alexa568 conjugated secondary was completed first. Then, the sections washed repeatedly and stained with Alexa647 conjugated ki67. Co-localization of eGFP+ astrocytes with ki67 and p-smad1/5 at P3 was examined in 3 independent brains. P-smad1/5 colocalization with eGFP+ astrocytes was examined in two independent mouse brains for each postnatal time point. Significance was determined using a students t-test.

Scholze A.R., Foo L.C., Mulinyawe S, & Barres B.A. (2014). BMP Signaling in Astrocytes Downregulates EGFR to Modulate Survival and Maturation. PLoS ONE, 9(10), e110668.

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Immunofluorescence Staining of Myoblasts

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Tissues (7 μm) and cells were fixed with 4% paraformaldehyde (PFA, Polysciences Europe GmbH) in PBS and permeabilized with 0.2% Triton X-100 in PBS containing 1% (w/v) bovine serum albumin (BSA). A blocking solution containing donkey serum (1:10 dilution in PBS; VWR) was applied, and primary antibodies (reported hereafter) were incubated overnight in PBS supplemented with 1% BSA. Secondary Alexa Fluor donkey antibodies were diluted at 4 μg/ml in PBS supplemented with 1% BSA, and the nuclei were counterstained with 10 μg/ml Hoechst. Fluor Save reagent (Millipore, Chemicon) was used as mounting medium. Images were acquired using an Eclipse Ti inverted microscope (Nikon). The fusion index (e.g. the number of myotube nuclei vs. the total number of nuclei) for hMAB cultures or the percentage of nuclei in GFP⁺ myotubes vs. the total number of nuclei for all co-cultures was calculated for a minimum of four random fields in each condition (n = 3).
The primary antibodies and their dilutions were as follows: 1:300 rabbit anti-GFP (Life Technologies) or 1:500 goat anti-GFP (Abcam; depending on the specific use for IF or WB analyses and on the combination with other antibodies), 1:3 mouse anti-MyHC (Developmental Studies Hybridoma Bank, DSHB, clone MF20), 1:1000 rabbit anti-P-SMAD1/5/8 or rabbit anti-P-SMAD1/5 (Cell Signalling), 1:200 rabbit anti-ID1 (Santa Cruz Biotechnology).

Costamagna D., Quattrocelli M., van Tienen F., Umans L., de Coo I.F., Zwijsen A., Huylebroeck D, & Sampaolesi M. (2015). Smad1/5/8 are myogenic regulators of murine and human mesoangioblasts. Journal of Molecular Cell Biology, 8(1), 73-87.

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