Anti mtor and p mtor

Lentivirus-infected HCT116 cells were collected, washed with ice-cold PBS and lysed in 2 × SDS sample buffer (100 mM Tris-HCl (pH 6.8), 10 mM EDTA, 4% SDS, 10% glycine). Samples containing equal amounts of protein (30 μg) were run on a 10% SDS-PAGE gel at 50 V for 3 h. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 1.5 h. Membranes were blocked using 1% BSA in TBST at RT for 1 h and then incubated with the primary antibodies anti-HOXC6 (1:100, Novus, NB100-92309), autophagy markers LC3A/B, Atg5, and Atg7 (1: 1000, Cell Signaling Technology, Autophagy Antibody Sampler Kit #4445), anti-SQSTM1/p62 (1:500, Cell Signaling Technology, #8025) or anti-mTOR and p-mTOR (1:1000, Cell Signaling Technology, #9964S) overnight at 4°C. After washing in TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (1:5,000, Santa Cruz, SC-2005) and goat anti-rabbit (1:5,000, Santa Cruz, SC-2054) secondary antibodies for 1 h at RT. Membranes were washed in TBS-T and signals were detected using an enhanced chemiluminescence kit (Pierce, USA).

Western blot analysis was performed as described previously [20 (link)]. Blots were probed and incubated overnight at 4°C with the rabbit polyclonal IgG anti-AKT (sc–8312), anti-p-AKT (Thr 308, sc–135650), anti-p-p70S6k (sc–7984), anti-p-ERK1 (Thr202 sc–101760), anti-p-p38 (Thr180/Tyr182, sc–101759), mouse monoclonal anti-p-JNK (Thr183/Tyr 185, sc–81502) (Santa Cruz) and rabbit polyclonal IgG anti-mTOR and p-mTOR (S2448, Cell Signaling). A mouse anti-human monoclonal antibody recognizing the human ß-actin (A5441; Sigma-Aldrich) was used as control in all experiments. Data are expressed as mean±SD intensity of optical density.