mtor pathway antibody sampler kit  (Cell Signaling Technology Inc)

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Mechanosensitive Ion Channel Piezo1 Regulates Chondrocyte Homeostasis Through the PI3K / AKT / mTORC1 Pathway in Osteoarthritis

doi: 10.1111/jcmm.70734

Figure Lengend Snippet: Activation of the Piezo1 channel induces the PI3K/AKT/mTORC1 pathway. (A) RNA sequencing was performed on chondrocytes treated with DMSO or 10 μM Yoda1 for 2 h, and comparative analysis of the data was conducted ( n = 3). The bar graph illustrates the most significantly up‐regulated biological processes in the GO functional enrichment analysis. (B) Bubble plots demonstrating significantly altered signalling pathways in Reactome enrichment analysis. (C–F) After treatment of chondrocytes with 10 μM Yoda1 for 0, 2, 6 and 12 h, western blot analysis was used to assess expression of p‐PI3K, PI3K, p‐AKT, AKT, p‐mTOR, mTOR and RAPTOR, and protein bands were quantified ( n = 3). (G, H) The levels of p‐PI3K, PI3K, p‐AKT, AKT, p‐mTOR, mTOR and RAPTOR expression were analysed by western blot after siNC and si Piezo1 ‐treated chondrocytes ( n = 3). All data were presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4°C with primary antibodies targeting Piezo1 (1:500, 15939–1‐AP, proteintech), GAPDH (1:1000, ET1601‐4, Huabio), COL2 (1:1000, GTX100829, Genetex), MMP13 (1:1000, 18165–1‐AP, proteintech), MMP3 (1:1000, ER1706‐77, Huabio), ACAN (1:1000, DF7561, affbiotech), ADAMTS5 (1:1000, ab41037, abcam), Phospho‐PI3K p85 alpha (Tyr607) (1:1000, AF3241, affbiotech), PI3K (1:1000, ET1608‐70, Huabio), Phospho‐Akt (Ser473) (1:1000, #4060, cell signalling technology), AKT (1:1000, ET1609‐47, Huabio), and mTOR pathway kit (1:1000, #9964, cell signalling technology).

Techniques: Activation Assay, RNA Sequencing, Functional Assay, Western Blot, Expressing