Pi3k and phosphorylated pi3k

Proteins were separated on SDS polyacrylamide gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with specific primary antibodies, including Akt, phosphorylated-Akt, COX-2, IκB-α, phosphorylated-IκB-α, p65, and Lamin B1 (Santa Cruz, CA, USA); ERK1/2, phosphorylated-ERK 1/2, p38, phosphorylated-p38, JNK, and phosphorylated-JNK (Millipore); PI3K and phosphorylated-PI3K (Cell Signaling Technology, Inc., MA, USA); and ICAM-1 and β-actin (Sigma). The membrane was washed and incubated with secondary antibodies for 1 h. Finally, specific proteins were detected with Luminol/Enhancer Solution (Millipore) using the BioSpectrum 600 system (UVP, Upland, CA, USA).

Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Filters were probed with the following specific primary antibodies: anti-CTSB (Cell signaling, Danvers, MA, USA), MMP-9 (Abcam, Cambridge, MA, USA), β-actin (Sigma, St Louis, MO, USA), PI3K and phosphorylated-PI3K (Cell signaling, Danvers, MA, USA), Akt and phosphorylated-Akt (Cell signaling, Danvers, MA, USA), mTOR, and phosphorylated-mTOR (Cell signaling, Danvers, MA, USA). Blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA) and visualized by chemiluminescence. The band density was quantified by densitometry using Scion Image software, and normalized to β-actin levels.