Rabbit anti pmad

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-pMad is a primary antibody that specifically binds to phosphorylated Mothers against decapentaplegic homolog (pMad), a key downstream effector of the transforming growth factor-beta (TGF-β) signaling pathway. This antibody can be used to detect and quantify the levels of activated pMad in various experimental systems.

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Lab products found in correlation

Lsm 780 confocal microscope, by Zeiss (3 mentions) Tcs sp2 aobs confocal microscope, by Leica (2 mentions) Mouse anti α tubulin, by Merck Group (1 mentions) Mouse anti egfr, by Abcam (1 mentions) Rabbit anti β galactosidase, by Promega (1 mentions) Goat anti mouse dylight 488, by Agrisera (1 mentions) Goat anti mouse dylight 549, by Agrisera (1 mentions) Rhodamine phalloidin, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) M3 medium, by Merck Group (1 mentions) Mouse anti patched, by Developmental Studies Hybridoma Bank (1 mentions) Rat anti ci 2a1, by Developmental Studies Hybridoma Bank (1 mentions) Mouse anti engrailed, by Developmental Studies Hybridoma Bank (1 mentions) Glycerol, by Merck Group (1 mentions) Hoechst, by Cell Signaling Technology (1 mentions) A1plus confocal microscope, by Nikon (1 mentions) Mouse anti β galactosidase, by Promega (1 mentions)

6 protocols using rabbit anti pmad

Imaging and Quantification of Drosophila Wing Disc

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Dissected wing imaginal discs were fixed and stained with antibodies according to standard procedures. The following primary antibodies were used: rabbit anti-pMad, 1:200 (Cell Signalling); mouse anti-α-Tubulin, 1:2000 (Sigma); rabbit anti-β-galactosidase, 1:2000 (Promega); mouse anti-Ubx, 1:200 (DSHB); rabbit anti-Omb, 1:1000 and rabbit anti-Sal, 1:500 (a gift from Coralia Pérez Fernández); mouse anti-EGFR, 1:200 (abcam); and mouse anti-Wg 1:200 (DSHB). Secondary antibodies (diluted 1:200) included goat anti-mouse DyLight 488 and goat anti-mouse DyLight 549 (Agrisera) and goat anti-rabbit DyLight Cy5 (Jackson ImmunoResearch). Cell nuclei were stained with DAPI (1:500, Sigma). F-actin was visualized with Rhodamine-phalloidin, 1:2000 (Sigma). Images were collected using a Leica TCS SP2 AOBS confocal microscope. Cell height was calculated based on high resolution confocal images using the Image-J program.

Tang W., Wang D, & Shen J. (2016). Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis. Scientific Reports, 6, 30236.

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Immunofluorescence Staining of Drosophila Wing Discs

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Discs were dissected from wandering third instar larvae and immunofluorescence staining was carried out as previously described [33 (link)]. Briefly, larvae are dissected in cold PBS (pH7.4) and fixed in 4% formaldehyde in PBS for 15 minutes. Samples were then permeabilized in PBS + 0.1% Triton X-100 and blocked in 5% normal horse serum. Primary antibodies in 5% normal horse serum were incubated overnight at 4°C or 2 hours at RT. Primary antibodies used for the immunostainings were mouse or rabbit α-V5 (Life Technology), rabbit anti-pMad (1:200) (Cell Signaling), rabbit α-Spalt major (1:100), anti-hedgehog (1:40), and chicken α-lacZ (1:100). Mouse anti-patched (DSHB), mouse anti-engrailed (DSHB), rat anti-Ci 2A1 (DSHB) were obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Department of Biological Sciences. Fluorophore conjugated secondary antibodies were from Jackson Immunoresearch. Confocal images were collected as single frames on a Zeiss LSM 780 confocal microscope. MG132 (100 μM; Calbiochem) in M3 medium (Sigma) was used to treat wing discs for up to 6 hours before immunostaining.

Wang G., Tang X., Chen Y., Cao J., Huang Q., Ling X., Ren W., Liu S., Wu Y., Ray L, & Lin X. (2014). Hyperplastic Discs Differentially Regulates the Transcriptional Outputs of Hedgehog Signaling. Mechanisms of development, 133, 117-125.

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Immunohistochemical Analysis of Drosophila

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The antibodies used were rabbit anti-Spalt (a kind gift from R. Barrio, [47 (link)]), rabbit anti-pMAD (a kind gift from G. Morata), rabbit anti–phosphorylated histone 3 (PH3) (Cell Signaling), rabbit anti-βgal (Cappel), rat anti-Ci (2A1, DSHB), and mouse anti-Dally-like (13G8, DSHB). Secondary antibodies were obtained from Molecular Probes. TUNEL analysis was performed as described in [74 (link)].

Ferreira A, & Milán M. (2015). Dally Proteoglycan Mediates the Autonomous and Nonautonomous Effects on Tissue Growth Caused by Activation of the PI3K and TOR Pathways. PLoS Biology, 13(8), e1002239.

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Immunostaining Identification of Germline Stem Cells

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The expression of pMad is highly specific for GSCs [24 (link)]. Therefore, we used rabbit anti-pMad (1:400; Cell Signaling Technology) as a primary antibody to label the GSCs in the germarium of the ovariole. Further, we differentiated CBs with the differentiating cysts based on the presence of round spectrosomes. For this purpose, we used mouse anti-α-Spectrin (3A9, 1:100; Developmental Studies Hybridoma Band [DSHB]) as a primary antibody (Figure 2).

Khalid M.Z., Sun Z., Chen Y., Zhang J, & Zhong G. (2022). Cyromazine Effects the Reproduction of Drosophila by Decreasing the Number of Germ Cells in the Female Adult Ovary. Insects, 13(5), 414.

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Ovarian Antibody Staining Protocol

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Antibody staining was performed as previously described (Liu et al., 2015 (link)). In brief, the ovaries were first fixed in 4% paraformaldehyde (PFA), washed three times with PBST (0.1% Triton X-100 in PBS), blocked in 5% normal goat serum (NGS), and incubated with primary antibodies at 4°C overnight. The next day, ovaries were washed with PBST three times, blocked in 5% NGS, incubated with secondary antibodies for 2 h, and washed with PBST three times. In addition, Hoechst (1:5,000; Cell Signaling Technology, Danvers, MA, United States) was used to stain the DNA. The samples were then mounted in 90% glycerol (Sigma).
Following primary antibodies were used: rabbit anti-pMad (1:400; cell signaling), mouse anti-α-Spectrin [3A9, 1:100; Developmental Studies Hybridoma Band (DSHB)], and rabbit anti-vasa (1:3,000). Secondary antibodies used were rabbit cy3 (1:1,000) and mouse 488 (1:1,000). Nikon A1 plus confocal microscope was used to take images (Nikon, Tokyo, Japan).

Khalid M.Z., Sun Z., Zhang J., Zhang S, & Zhong G. (2022). Cyromazine affects the ovarian germ cells of Drosophila via the ecdysone signaling pathway. Frontiers in Physiology, 13, 992306.

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Immunostaining of Drosophila Imaginal Discs

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Fixed wing imaginal discs were stained with antibodies according to standard procedures. The primary antibodies used were: rabbit anti-Doc2, 1:2000 [30 (link)]; rabbit anti-pMad, 1:200 (Cell Signaling Technology #9516, Danvers, MA, USA); mouse anti-β-galactosidase, 1:2000 (Promega Z378B, Madison, WI, USA); Secondary antibodies used were goat anti-mouse DyLight 549, 1:200 (Agrisera AS09 643, Vännäs, Sweden) and goat anti-rabbit DyLight 488 1:200 (Agrisera AS09 637, Vännäs, Sweden). Images were collected using a Leica TCS-SP2-AOBS confocal microscope.

Lu J., Wang Y., Wang X., Wang D., Pflugfelder G.O, & Shen J. (2022). The Tbx6 Transcription Factor Dorsocross Mediates Dpp Signaling to Regulate Drosophila Thorax Closure. International Journal of Molecular Sciences, 23(9), 4543.

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