Alexa fluor 488 goat polyclonal anti rabbit igg

The antibodies used in the Western blot analysis were: mouse monoclonal anti-human β-actin, mouse monoclonal anti-human α-tubulin, mouse monoclonal anti-human E-Cadherin, mouse monoclonal anti-human p-GSK (T279/T216), mouse monoclonal anti-human p120 Catenin, and mouse monoclonal anti-human GLUT3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), rabbit monoclonal anti-human p-Src (pY416), rabbit monoclonal anti-human β-Catenin, rabbit monoclonal anti-human p-β-Catenin (S33/37/T41), rabbit monoclonal anti-human RhoA, and rabbit monoclonal anti-human p-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit monoclonal anti-human p-p120 catenin (pY228) from BD Biosciences (Franklin Lakes, NJ, USA). For fluorescence microscopy, the following were used as primary antibodies: rabbit monoclonal anti-human RhoA (Cell Signaling Technology); as secondary antibody Alexa Fluor^® 488 goat polyclonal anti-rabbit IgG (Jackson ImmunoResearch, Cambridge, UK) and DyLight^® 594 goat polyclonal anti-mouse IgG (Abcam, Cambridge, UK).

Cells were seeded on coverslips in 24-well culture plates at a density of 1 × 10⁵ cells/cm² and treated with iPA for 24 h at 37 °C. After, were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked using PBS-BSA 0.4%. Then, cells were incubated with primary antibody at 4 °C overnight. Following washes with PBS 1× for three times, cells were incubated with a labeled secondary antibody at room temperature for 1 h. Nuclei were then stained with DAPI (Hoechst, Life Technologies Corporation). Finally, cells were washed with PBS 1× for three times and mounted on the slide using Dako Fluorescent Mounting Medium. The images were acquired using a Leica Thunder Imaging System (Leica Microsystems Srl, Buccinasco, Italy) equipped with Leica DFC9000GTC camera and a planapo ×100 oil immersion (NA 1.4) objective lens. Images were acquired by using the Small Volume Computational Clearing (SVCC) mode. For fluorescence microscopy, the following were used as primary antibody: mouse monoclonal anti-human RIP3 (Santa Cruz Biotechnology, CA, USA; dilution 1:200), rabbit monoclonal anti-human pMLKL (Abcam, Cambridge, UK; dilution 1:200); secondary antibody: Alexa Fluor® 488 goat polyclonal anti-rabbit IgG (Jackson ImmunoResearch Europe Ltd., Cambridge, UK; dilution 1:200), DyLight® 594 goat polyclonal anti-mouse IgG (Abcam, Cambridge, UK; dilution 1:200).