Anti p p70s6k thr389

Cholangiocellular carcinoma cells were cultured in serum-free DMEM. Supernatant of OSI-027 treated cells was collected. Equal amounts of protein (4 µg of supernatant, 20 µg of lysate) were applied on 10% SDS-polyacrylamide gels and electrophoresed (BioRad, Munich, Germany) as described before (Guenzle et al., 2017 (link)). Blots were incubated with primary antibodies anti-MMP2 (#4022 1:1000 Cell Signaling Technology), anti-AKT (#9272 1:1000 Cell Signaling Technology), anti-pAKT^Ser473 (#9271 1:1000 Cell Signaling Technology), anti-ERK1/2 (#4696 1:1000 Cell Signaling Technology), anti-pERK^{Thr202/Tyr204} 1/2 (#4370 1:1000 Cell Signaling Technology), anti-p38 (#8690 1:1000 Cell Signaling Technology), anti-p-p38^{Thr180/Tyr182} (#4511 1:1000 Cell Signaling Technology), anti-4EBP1^Ser65 (#9451 1:1000 Cell Signaling Technology), anti-p-p70S6K^Thr389 (#9206 1:1000 Cell Signaling Technology), anti-pSTAT3^Tyr705 (#9145 1:1000 Cell Signaling Technology), anti-PDI (sc-74551 1:1000 Santa Cruz). Proteins were visualized by enhanced chemiluminescence (BioRad, Munich, Germany). PDI was used as loading control for the whole cell lysate. Finally, densitometry of the presented western blot was performed using ImageJ. Ratio was calculated to pixel/area of reference PDI. Expression of phosphorylated protein was calculated in relation to total protein amount.

Total protein from cell death and autophagy assays was analyzed by western blot as previously described [25 (link)]. The following antibodies were used: Anti-LC3A/B (1:1000), anti-SQSTM1/p62 (1:1000), anti-p-p70^S6K (Thr389) (1:1000), anti-p-4EBP1 (1:1000), anti-p-mTOR (Ser2448) (1:1000), α-tubulin (1:2000), and β-actin (1:2000), all purchased from Cell Signaling (Danvers, MA, USA). β-actin and α-tubulin were used as a loading control. HRP-conjugated goat anti-mouse and goat anti-rabbit (all from Cell Signaling) were used as secondary antibodies. Chemiluminescence using ECL (GE Healthcare Life Sciences) was detected on an Image Quant LAS4000 mini photo documentation system (GE Healthcare Life Sciences). The subsequent quantification was performed by Image J software version 1.52s (National Institutes of Health—https://imagej.nih.gov/ij/).

Total proteins from tissues were separated by SDS-PAGE, blotted and probed with anti-Akt (Cell Signaling, Danvers, MA, USA), anti-phospho-Akt (ser473; Cell Signaling), anti-β-actin antibody (Santa Cruz, CA, USA), anti-Lin28a (Abcam, Cambridge, MA, USA), anti-p70S6k (Cell Signaling), anti-p-p70S6k (Thr389, Cell Signaling), anti-Cleaved Caspase-3(Sigma-Aldrich), anti-Caspase-3 (Sigma-Aldrich), anti-Bcl-2 (Sigma-Aldrich), anti-Bax (Sigma-Aldrich). The Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify protein concentrations. The blots were visualized with a chemiluminescence system (Amersham Bioscience, Buchinghamshire, UK). The signals were quantified by densitometry.

All reagents were from Sigma (St Louis, MO, USA) unless otherwise indicated.
For immunochemical analysis, antibodies against Vav1 (sc‐132), Akt1 (sc‐1618), Akt2 (sc‐5270), Akt3 (sc‐11520), p‐Akt1/2/3 (sc‐14032), Bcl‐2 (sc‐509), Caspase‐3 (sc‐371), and IkBα (sc‐7148) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐p‐Akt1 (Ser473) (#4060), anti‐p‐Akt2 (Ser474) (#8599), anti‐p‐P70S6K (Thr389) (#9205), and anti‐P70S6K (#9202) were from Cell Signaling Technology (Danvers, MA, USA). Anti‐Bax (#610983) was from BD Biosciences (Milan, Italy), anti‐Cyclin D1 (#04‐1151) was from Merck Millipore (Milan, Italy), and anti‐β‐Tubulin (#T4026) was from Sigma.
For immunohistochemical analysis, the anti‐Vav1 (sc‐132) and the anti‐Akt1 (sc‐377457) antibodies were from Santa Cruz Biotechnology, anti‐p‐Akt (Ser473) (#3787) antibody was from Cell Signaling Technology, and the anti‐Cyclin D1 (MCP511) antibody was from YLEM (Rome, Italy).

The following primary antibodies were used for the western blotting: anti‐p70S6K (#9202; Cell Signaling Technology), anti‐p‐p70S6K (Thr389, #9205; Cell Signaling Technology), anti‐rpS6 (#2217; Cell Signaling Technology), anti‐p‐S6 (Ser240/244, #5364P; Cell Signaling Technology), anti‐eIF4E‐binding protein 1 (4EBP1) (#9452; Cell Signaling Technology), anti‐p‐4EBP1 (Thr37/46, #2855S; Cell Signaling Technology), anti‐extracellular signal‐regulated kinase 1/2 (ERK1/2) (#9102; Cell Signaling Technology), anti‐p‐ERK1/2 (Thr202/Tyr204, #9101; Cell Signaling Technology), anti‐p38 MAPK (p38) (#9212; Cell Signaling Technology), and anti‐p‐p38 (Thr180/Tyr182, #9211; Cell Signaling Technology).