Goat anti mouse igg

Rictor and mTOR complementary DNA were kind gifts from Dr X. F. Steven Zheng (Rutgers Cancer Institute of New Jersey). All constructs were subcloned into pcDNA3.1 with an N-terminal HA tag. A rabbit polyclonal antibody against NOP14 (NBP2-13665) was purchased from Novus. Antibodies against phospho-Akt (Ser473) (4060), phospho-AKT (Thr450) (9267), phospho-Akt (Thr308) (9275), Akt (9272), phospho-mTOR (Ser2448) (5536), mTOR (2983), Rictor (9476), Sin1 (12860), phospho-S6K (Thr389) (9205), phospho-4EBP1 (Thr37/46) (2855); FoxO1 (2880), ribosomal protein S6 (2317), phospho-S6 (Ser235/236) (2211), ribosomal protein L7 (2415), c-Myc (13987), HA-tag (2367), S-tag (EM50105), Myc-tag (EM31105), and β-actin (4970) were purchased from Cell Signaling Technology. Anti-KDEL (Alexa Fluor 488-conjugated) (ab184819) and anti-GAPDH (ab75479) antibodies were obtained from Abcam. Anti-tubulin (sc-23948) antibody was purchased from Santa Cruz Biotechnology. A mouse monoclonal antibody against FLAG (F3165) was obtained from Sigma‒Aldrich. Peroxidase–conjugated goat anti-rabbit IgG and goat anti-mouse IgG (Amersham Pharmacia Biotech) secondary antibodies were used for Western blotting analysis.
LY294002, AZD8055, and rapamycin were all purchased from Selleck Chemicals.

Cells were lysed using RIPA lysis buffer (Beyotime, China), and the protein concentrations were measured using the Bicinchoninic Acid Protein Assay Kit (Beyotime, China). The proteins were separated on a 10% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany), as described previously^{28 (link)}. Following 2 h of blocking with TBST supplemented with 5% non-fat dried milk, the membranes were incubated overnight with specific primary antibodies at 4 °C. Anti-PI3K (1:1000, Abcam), anti-Bcl-2 (1:1000, Abcam), anti-AKT (1:2000, Abcam), anti-pAKT (1:500, Abcam), anti-BAX (1:1000, Abcam), anti-LC3B-I (1:500, Abcam), anti-p62 (1:2000, Abcam), anti-mTOR (1:1000, Abcam), anti-cleaved caspase 3 (1:1000, Abcam), and anti-pro-caspase 3 (1:1000, Abcam) were used as primary antibodies. A next step involved washing the membranes, exposing them to peroxidase-conjugated secondary antibodies, and developing them with ELC (Amersham Pharmacia, UK); incubation at room temperature for another 60 min was performed with goat anti-rabbit IgG (1:5000) or goat anti-mouse IgG (1:5,000). Thermo provided the software and hardware for measuring immunoreactivity. The Super Signal West Femto Maximum Sensitivity Substrate Kit was used on a C-Digit Blot Scanner to measure immunoreactivity.