After the indicated duration of treatment, cells were lysed in phosphate buffered saline (PBS) containing 50mM Tris pH 8.0, 1% igepal, 0.1% SDS, 0.5% sodium deoxycholate and Roche complete protease inhibitor cocktail (Roche, Basel, Switzerland). Lysates were sonicated and frozen at −80°C until used. Cell lysates (20–40μg total protein) were separated on reducing SDS-PAGE gels and proteins were transferred to polyvinylidene fluoride membranes by semi-dry blotting. Membranes were then blocked and probed with rabbit polyclonal antibodies towards JNK (catalogue # 9252), phospho-JNK (catalogue # 9251), Smad1 (catalogue # 9743), phospho-Smad1/5/8 (catalogue # 9511, all Cell Signaling Technology, Danvers, MA) or PCNA (catalogue # ab18197, Abcam, Cambridge, UK), rabbit monoclonal antibodies towards Caspase-3 (clone 8G10), cleaved Caspase-3 (clone 5A1E), phosphorylated Smad1/5 (clone 41D10, all Cell Signaling Technology, Danvers, MA), Smad5 (clone EP619Y, Epitomics, Burlingame, CA), Id1 (clone 195-14) or Id3 (clone 17-3, both CalBioreagents, San Mateo, CA) or a mouse monoclonal antibody towards BMPR-II (clone 18/BMPR-II, BD Transduction Laboratories, Franklin Lakes, NJ). As a loading control, all blots were re-probed with a monoclonal antibody towards either α-tubulin (clone DM1A) or β-actin (clone AC-15, Sigma). Densitometry was performed using ImageJ software.
Smad5 clone ep619y
Manufactured by Abcam
Smad5 (clone EP619Y) is a rabbit monoclonal antibody that recognizes the Smad5 protein. Smad5 is a member of the Smad family of proteins, which are key mediators of transforming growth factor-beta (TGF-β) signaling. The Smad5 antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunofluorescence, to detect and study the Smad5 protein.
Automatically generated - may contain errors
Lab products found in correlation
β actin clone ac 15, by Merck Group (2 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
Smad1, by Cell Signaling Technology (2 mentions)
Phospho smad1 5 8, by Cell Signaling Technology (2 mentions)
Clone 18 bmpr 2, by BD (2 mentions)
α tubulin clone dm1a, by Merck Group (2 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
2 protocols using smad5 clone ep619y
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of Signaling Pathways
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After the indicated duration of treatment, cells were lysed in phosphate buffered saline (PBS) containing 50mM Tris pH 8.0, 1% igepal, 0.1% SDS, 0.5% sodium deoxycholate and Roche complete protease inhibitor cocktail (Roche, Basel, Switzerland). Lysates were sonicated and frozen at −80°C until used. Cell lysates (20–40μg total protein) were separated on reducing SDS-PAGE gels and proteins were transferred to polyvinylidene fluoride membranes by semi-dry blotting. Membranes were then blocked and probed with rabbit polyclonal antibodies towards JNK (catalogue # 9252), phospho-JNK (catalogue # 9251), Smad1 (catalogue # 9743), phospho-Smad1/5/8 (catalogue # 9511, all Cell Signaling Technology, Danvers, MA) or PCNA (catalogue # ab18197, Abcam, Cambridge, UK), rabbit monoclonal antibodies towards Caspase-3 (clone 8G10), cleaved Caspase-3 (clone 5A1E), phosphorylated Smad1/5 (clone 41D10, all Cell Signaling Technology, Danvers, MA), Smad5 (clone EP619Y, Epitomics, Burlingame, CA), Id1 (clone 195-14) or Id3 (clone 17-3, both CalBioreagents, San Mateo, CA) or a mouse monoclonal antibody towards BMPR-II (clone 18/BMPR-II, BD Transduction Laboratories, Franklin Lakes, NJ). As a loading control, all blots were re-probed with a monoclonal antibody towards either α-tubulin (clone DM1A) or β-actin (clone AC-15, Sigma). Densitometry was performed using ImageJ software.
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