Sds page gels

The stimulated cancer cell lines were lysed with radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Danfoss, MA, USA) containing phenylmethanesulfonyl fluoride (Beyotime). Equal amounts of protein lysate were separated on 10% or 12.5% SDS PAGE gels (Abcam, Cambridge, UK) and then transferred to polyvinylidene fluoride membranes (EMD Millipore). The membranes were incubated with the primary antibodies at 4°C overnight. The following primary antibodies were used: anti-human matrix metallopeptidase 2 (MMP2) (#4022; Cell Signaling Technology), MMP9 (#13667; Cell Signaling Technology), p-ERK (4370P; Cell Signaling Technology), extracellular signal-regulated kinase 1/2 (ERK1/2) (4695P; Cell Signaling Technology), Akt (#4691; Cell Signaling Technology), p-Akt (#4060; Cell Signaling Technology), Bcl-2 (#4223; Cell Signaling Technology), Bax (#2772; Cell Signaling Technology), p27 (#2552; Cell Signaling Technology), p-mTOR (#2971; Cell Signaling Technology), mTOR (2983P; Cell Signaling Technology), cyclinD1 (#2978; Cell Signaling Technology), and GAPDH (AF0009; Beyotime). After incubation, the membranes were incubated with an horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Then, the membranes were washed six times with TBST (PBS with 0.05% Tween20). The blot bands were visualized by Find-do ×6 Tanon (Tanon, Shanghai, China).

TLysis buffer (Cell Signaling Technology, Danfoss, MA, USA) containing phenylmethanesulfonyl fluoride (Beyotime) was used to lyse the stimulated cancer cell lines. Equal amounts of extracts were separated to 10% or 12.5% SDS PAGE gels (Abcam, Cambridge, UK), transferred onto polyvinylidene fluoride membranes (EMD Millipore), followed by incubation with primary antibodies at 4°C overnight. The primary antibodies were anti-CDC6 (ab109315, abcam), Ki-67 (ab245113, abcam), anti-PCNA (ab92552, abcam) and GAPDH (AF0009; Beyotime). HRP-conjugated secondary antibodies were used to incubate the membranes for 1 hour at room temperature, after which they were washed with TBST (PBS with 0.05% Tween20) 6 times. Finally, the visualization of the blot bands was achieved by Find-do ×6 Tanon (Tanon, Shanghai, China).