Phospho s6 ribosomal protein

IFN-γ, Doxycycline and rapamycin were purchased from Sigma-Aldrich Corporation (Saint Louis, MO). Antibodies to detect mTOR, phospho-mTOR, p70 S6 kinase, phospho-p70 S6 kinase, S6 ribosomal protein, and phospho-S6 ribosomal protein were purchased from Cell Signaling (Danvers, MA). PD-L1 antibody was purchased from Abcam (Cambridge MA). The TUSC2 polyclonal antibody was developed in Bethyl Laboratories (Montgomery, TX). Lipofectamine 2000 was purchased from Invitrogen. Nanoparticle–TUSC2 complexes were made as previously described [29 (link)].

HeLa cells or 293T cells were seeded in 12-well plate (2 × 10⁵ cells/ml) 24 hours before each experiment. Immediately after the indicated treatments, cells were lysed using 120 μL of 2 x loading buffer (50 mM Tris pH 6.8, 10% glycerol, 2% SDS, 0.05% bromophenol blue and 100 mM DTT), boiled at 95°C for 5 minutes and sonicated. 10 μL of extract were loaded on a 4%–12% Bolt Bis-Tris Plus gel (ThermoFisher Scientific).
Immunoblots were visualized using the Biorad ChemiDoc™ touch imaging system (Biorad).
The following antibodies were used: CHOP (Thermo Fisher Scientific, MA1-250, 1/500 dilution), p-eIF2α (ThermoFisher Scientific, 44-728G, 1/1000 dilution), tubulin (Sigma-Aldrich, T5168, 1/2000 dilution), MRPL44 (Proteintech, 16394-1-AP, 1/1000 dilution), MRPL28 (Abcam, ab126719, 1/1000 dilution), RPL4 (Proteintech, 11302-1-AP, 1/1000 dilution), RPS6 (Cell Signaling, 2217S, 1/1000 dilution), HSPC014 (Abcam, ab170865, 1/1000 dilution) phospho-S6 ribosomal protein (Cell Signaling, 4856S, 1/1000 dilution) and ATF4 (Santa Cruz Biotechnology, sc-390063, 1/1000 dilution).

To prepare whole-cell lysates, cells were washed twice with cold PBS and lysed in M-PER Mammalian Protein Extraction Reagent (product no. 78501, Thermo Fisher Scientific) supplemented with Pierce protease and phosphatase inhibitor cocktail (product no. A32961, Thermo Fisher Scientific). Tumor tissue samples were lysed in T-PER Tissue Protein Extraction Reagent (product no. 78510, Thermo Fisher Scientific) with above supplement. The automatic hand-operated OMNI-TIP Homogenizer (Omni International, Inc.) was used to homogenize the tumor tissues. Lysates from whole cells and tumor homogenates were then processed for SDS-PAGE Western blotting. The following antibodies were used: Phospho-MEK1/2 (S217/221; catalog no. 9154S), MEK1/2 (catalog no. 4694S), Phospho-AKT (S473; catalog no. 9271S), Phospho-AKT (T308; catalog no. 4056S), AKT (catalog no. 9272S), Phospho-Erk1/2 (catalog no. 4370S), Erk1/2 (catalog no. 4695S), Phospho-S6 Ribosomal Protein (Ser 235/236; catalog no. 4858S), S6 Ribosomal Protein (catalog no.2217S), cleaved-CASP-3 (catalog no. 9664), cleaved-PARP (catalog no. 9541) from Cell Signaling Technology; anti-β-ACTIN (catalog no A5441-.2ML from Sigma-Aldrich; KRAS (catalog no. OP24, from EMDMillipore), and RAF1 (catalog no. sc-7267, Santa Cruz Biotechnology), horseradish peroxidase–conjugated goat anti-GST antibody (catalog no. A190-121P from Bethyl Laboratories).

Cells were lysed in buffer containing 60 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, and 20% (v/v) glycerol, and protein quantitated by BCA assay. Cell lysates containing 25 μg of protein were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-S6 ribosomal protein (Ser240/244) (Cell Signalling Cat. No. 2215) (1:1000 for anti-phospho RPS6), total RPS6 (Cell Signalling Cat. No. 2217) and β-actin (1:5000) (Sigma). Protein bound primary antibody was subsequently incubated with respective secondary antibody prior to membrane exposure to SuperSignal West Pico (Thermo Scientific) for β -actin or ECL plus for phospho-RPS6 (Thermo Scientific). Resulting bands were detected using chemiluminescence detection system (Fujifilm Las-3000).