Magic™ Anti-SMAD3 (C-terminal)(phospho S423) polyclonal antibody

Doxorubicin (DOX) is widely used clinically for the treatment of various malignant tumors, but it is accompanied by severe body toxic reactions, especially cardiac toxicity. This article explores the effects of pilose antler peptide (PAP) on DOX-induced myocardial injury and related mechanisms through in vitro experiments. Different concentrations of DOX were used to treat H9c2 cells for 6, 12, 24, and 48 h, respectively, to determine the IC50. Different concentrations of PAP were treated to H9c2 cells for 24, 48, and 72 h to determine the effect of PAP on H9c2 cells. The experiment was divided into control group, DOX group, PAP group and DOX+PAP group. Lactate dehydrogenase (LDH) leakage and creatine kinase MB (CK-MB) level were used to detect cell viability, flow cytometry was used to detect cell cycle and apoptosis, and immunofluorescence was used to detect Bax and Bcl-2 expression. The protein levels of TGF-beta / Smad / ERK signaling pathway were detected by Western blot. We found that PAP significantly increased cell viability after DOX-induced injury, reduced LDH, CK-MB levels, up-regulated Bcl-2 expression and down-regulated Bax expression levels. In addition, PAP can delay cell G2/M phase arrest, reduce apoptosis, significantly reduce TGF-beta(1) protein levels and Smad2, Smad3, ERK phosphorylation levels. These results suggest that PAP can protect DOX-induced H9c2 damage by inhibiting the TGF-beta / Smad / ERK signaling pathway.

IL-33 has emerged as a central mediator of immune, inflammatory, and fibrotic responses. Many studies have focused on mature IL-33, but elevated expression of the precursor, full-length IL-33 (FLIL33), has also been implicated in a spectrum of diseases, including tissue fibrosis. We previously reported and now confirmed that overexpression of FLIL33 induced phosphorylation of the key profibrotic signaling mediator of TGF-beta, Smad3, in primary human lung fibroblasts from healthy donors and idiopathic pulmonary fibrosis patients. Presently, we demonstrate that FLIL33-induced Smad3 phosphorylation was not abrogated by anti-TGF-beta antibody but was abrogated by ALK5/TGFBR1-specific and Smad3-specific inhibition, indicating that FLIL33 effect was in- dependent of TGF-beta but dependent on its receptor, TGFBR. Western blotting analyses revealed that FLIL33 overexpression increased levels, but did not affect subcellular distribution, of the AP2A1 and AP2B1 subunits of the adaptor protein complex 2 (AP2), a known TGFBR binding partner. siRNA-mediated inhibition of these subunits blocked FLIL33-induced Smad3 phosphorylation, whereas AP2 subunit overexpression induced Smad3 phosphorylation even in the absence of FLIL33. RNA-Seq transcriptomic analyses revealed that fibroblast stimulation with TGF-beta induced major changes in expression levels of numerous genes, whereas overexpression of FLIL33 induced modest expression changes in a small number of genes. Furthermore, qRT-PCR tests demon- strated that despite inducing Smad3 phosphorylation, FLIL33 did not induce collagen gene transcription and even mildly attenuated TGF-beta-induced levels of collagen I and III mRNAs. We conclude that FLIL33 induces Smad3 phosphorylation through a TGF-beta-independent but TGF-beta receptor- and AP2- dependent mechanism and has limited downstream transcriptomic consequences.