Smad1 5 8

Manufactured by Abcam

Sourced in United States

Smad1/5/8 is a protein involved in the transforming growth factor-beta (TGF-β) signaling pathway. It functions as a transcriptional regulator that mediates cellular responses to TGF-β and related cytokines.

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Lab products found in correlation

Containingprotease and phosphatase inhibitors, by Merck Group (1 mentions) C fos, by Cell Signaling Technology (1 mentions) Nfkb1, by Cell Signaling Technology (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Ecl chemiluminescence reagent, by Solarbio (1 mentions) Runx2, by Abcam (1 mentions) P smad1 5 8, by Abcam (1 mentions) Bmpria, by Abcam (1 mentions) Bmprib, by Abcam (1 mentions) Bmprii, by Abcam (1 mentions) Hrp conjugated goat anti rabbit igg, by Abcam (1 mentions) Gapdh, by Abcam (1 mentions) Ripa lysis buffer, by Solarbio (1 mentions) Bca kit, by Solarbio (1 mentions) Sds page, by Solarbio (1 mentions) Electrochemiluminescence plus reagent, by Thermo Fisher Scientific (1 mentions) Erk1 2, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)

4 protocols using smad1 5 8

Protein Extraction and Western Blot Analysis

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Whole cells were extracted in radioimmunoprecipitation assay buffer containing
protease and phosphatase inhibitors (Sigma-Aldrich, USA). The protein concentration
was measured with a bicinchoninic acid protein assay kit following the manufacturer's
instructions. The proteins were resolved by SDS-PAGE, transferred to a polyvinylidene
fluoride membrane and incubated overnight at 4°C with primary antibodies against
phospho (P)-SMAD 1/5/8 (Cell Signaling, USA), SMAD 1/5/8 (Abcam, USA), NFKB1 (Cell
Signaling), c-Fos (Cell Signaling), p-P38 (Cell Signaling), p-JNK and p-IkB (Anbobio,
China). The same membranes were probed with anti-β-actin antibodies (Sigma) to
normalize protein loading in each lane. Subsequently, the blots were washed in
Tris-buffered saline with Tween (10 mM Tris-HCl, 50 mM NaCl, and 0.25% Tween-20) and
incubated with the secondary antibody. Immunoreactive bands were visualized using an
ECL Plus substrate (GE Healthcare Life Sciences, China).

Sun S.X., Guo H.H., Zhang J., Yu B., Sun K.N, & Jin Q.H. (2014). BMP-2 and titanium particles synergistically activate osteoclast formation. Brazilian Journal of Medical and Biological Research, 47(6), 461-469.

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Protein Expression Analysis in Cell Culture

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Total cellular protein was extracted in RIPA lysis buffer (Solarbio) and obtained by centrifugation at 4°C for 15 min at 12,000 rpm, and protein concentrations were quantified by a BCA kit (Solarbio). The samples were separated by 10% SDS‐PAGE (Solarbio) and transferred to a PVDF membrane. After being blocked with 5% skimmed milk in PBS for 2 h, the membrane was incubated separately overnight with the following rabbit antibodies at 4°C: acetylated α‐tubulin, γ‐tubulin, α‐tubulin, collagen type 1 (COL‐1), osterix (OSX), BMP‐2, RUNX‐2, BMPRII, BMPRIA, BMPRIB, p‐Smad1/5/8, Smad1/5/8, IFT88 or GAPDH (all from Abcam, 1:1000). After probing with a secondary antibody (goat anti‐rabbit HRP‐conjugated IgG at 1:1000, Abcam), immunoreaction signals were visualized with the ECL chemiluminescence reagent (Solarbio).

Liu J., Leng F., Gao Y., He W., Wang J., Xian C.J., Ma H, & Chen K. (2022). Protection of primary cilia is an effective countermeasure against the impairment of osteoblast function induced by simulated microgravity. Journal of Cellular and Molecular Medicine, 27(1), 36-51.

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Western Blot Analysis of hBMSCs Proteins

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Isolated total protein of hBMSCs from each group by Radio Immunoprecipitation Assay (RIPA) buffer with a concentration of 1 mM phenylmethanesulfonylfluoride fluoride and measured protein concentration by Bicinchoninic Acid (BCA) protein assay kit from Beyotime (Shanghai, China). Then, separated proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membranes (Bio-Rad, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies as GAPDH (1:1,000), extracellular regulated protein kinases 1/2 (ERK 1/2) (1:1,000) (Cell Signaling Technology/CST, USA), and Smad1/5/8 (1:1,000) (Abcam, the UK) at 4 °C overnight. The respective secondary antibodies were applied after incubation. The selected bands were cut and then detected with electrochemiluminescence plus reagent (Invitrogen, the USA).

Wang F., Ye Y., Zhang Z., Teng W., Sun H., Chai X., Zhou X., Chen J., Mou H., Eloy Y., Jin X., Chen L., Shao Z., Wu Y., Shen Y., Liu A., Lin P., Wang J., Yu X, & Ye Z. (2023). PDGFR in PDGF-BB/PDGFR Signaling Pathway Does Orchestrates Osteogenesis in a Temporal Manner. Research, 6, 0086.

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Immunofluorescent Characterization of Cilia

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ROBs were grown on coverslips and were fixed in 4% paraformaldehyde and then permeabilized with 0.2% Triton X‐100 (PBST) for 10 min. After a brief wash, they were incubated for 1 h in 1% bovine serum albumin in PBS. Immunofluorescent staining was carried out overnight at 4°C with primary antibodies against acetylated α‐tubulin, γ‐tubulin, BMP‐2, BMP receptors (BMPRII, BMPRIA and BMPRIB), Smad1/5/8 or phosphorylated (p)‐Smad1/5/8 (1:500, all from Abcam). After washes, the cells were probed with secondary antibodies conjugated with Alexa 594 or Alexa 488 (1:1000; Abcam) for 1 h at 37°C. Cellular nuclear DNA was stained with 4′‐6‐diamidino‐2‐phenylindole (DAPI) (1:800; Solarbio). The cells were imaged under Delta Vision Imaging System (Delta Vision Ultra; Cytiva). The lengths of primary cilia were measured with Image‐Pro Plus 6.0 software (Media Cybernetics). The average length of each group was calculated from at least 40 primary cilia (n = 40). The percentage of ciliated cells was obtained by counting the number of ciliated cells and the total number of cells in 10 different fields of view.

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