Cells were fixed in 4% paraformaldehyde, post‐fixed using absolute methanol for 5 min, and stained according to previously published protocols (Mattioli et al., 2011 ). Primary antibodies were applied overnight at 4°C, and secondary antibodies were used for 1 hour at room temperature. Nuclei were counterstained with 4,6‐diamidino‐2‐phenylindole (DAPI). Sample observation and image acquisition were performed using a Nikon Eclipse Ni epifluorescence microscope equipped with a digital CCD camera and NIS‐ Elements 4.3 AR software. Photoshop CS and Photoshop 7 were used for image processing. Mean fluorescence intensity (MFI) was measured using NIS‐ Elements 4.3 AR.
For Western blot analysis, tissues and cells were lysed in buffer containing: 20 mM Tris‐HCl (pH = 7.5), 1% SDS, 1 mM Na3VO4, 1 mM PMSF, 5% beta‐mercaptoethanol, and protease inhibitors. Proteins were subjected to SDS gradient gel (5%–20%) electrophoresis and transferred to nitrocellulose membrane overnight at 4°C. After incubation with primary and secondary antibodies, immunoblotted bands were revealed by Invitrogen ECL detection system. Densitometry was performed by a Bio‐Rad GS800 Densitometer equipped with Quantity One Software. Densitometric values were normalized to corresponding GAPDH bands if not differently stated.
For Western blot analysis, tissues and cells were lysed in buffer containing: 20 mM Tris‐HCl (pH = 7.5), 1% SDS, 1 mM Na3VO4, 1 mM PMSF, 5% beta‐mercaptoethanol, and protease inhibitors. Proteins were subjected to SDS gradient gel (5%–20%) electrophoresis and transferred to nitrocellulose membrane overnight at 4°C. After incubation with primary and secondary antibodies, immunoblotted bands were revealed by Invitrogen ECL detection system. Densitometry was performed by a Bio‐Rad GS800 Densitometer equipped with Quantity One Software. Densitometric values were normalized to corresponding GAPDH bands if not differently stated.