Phospho p70s6k

Flow cytometry sorted cells were washed in PBS and resuspended in RIPA buffer, 1 mM PMSF, 1 mM Na₃VO₄, and 1 × protease inhibitor cocktail for 3 min on ice. The lysate was centrifuged at 14,000 × g for 15 min at 4 °C, and the supernatant was used for western blotting. Protein lysates were boiled in loading buffer (Beyotime, Jiangsu, China), resolved by electrophoresis on 8% SDS-polyacrylamide gels, and transferred to PVDF membranes (Amersham Pharmacia Biotech, Amersham, UK). Membranes were probed overnight at 4 °C with primary antibodies recognizing ERK1/2, phospho-ERK (Thr202/Tyr204), AMPKα, phospho-AMPKα (Thr172), P70S6K, and phospho-P70S6K (Thr389) (Cell Signaling, Danvers, MA, USA), with GAPDH (Cell Signaling) as the control. Horseradish peroxidase-conjugated IgG (Beyotime) was used to detect specific proteins. Finally, immunodetection was conducted using chemiluminescent substrates (Amersham Pharmacia Biotech).

Western blot analysis was performed as previously reported 19. Briefly, cells were washed with cold phosphate‐buffered saline, and the proteins were extracted with ice‐cold lysis buffer (20 mm 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid, pH 7.4, 150 mm NaCl, 1% sodium dodecyl sulfate, and 1% nonyl phenoxypolyethoxylethanol (NP‐40)) and separated by 8–15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gels were transferred to polyvinylidene difluoride membranes (Immobilon‐P; Millipore, Billerica, MA, USA) and blocked in 5% (w/v) milk in Tris‐buffered saline with Tween 20. After blocking, the membranes were incubated with primary antibodies overnight at 4 °C and then with horseradish peroxidase‐conjugated secondary antibodies for 1 h at room temperature. The blots were enhanced with either Luminata™ Forte Western HRP Substrate (Millipore, USA) or SuperSignal West Pico chemiluminescence substrate (Thermo Scientific, Waltham, MA, USA), and detected using an Amersham Imager 600 (GE Healthcare, Tokyo, Japan). The antibodies used in this study were as follows: phospho‐Akt (Ser473), Akt, phospho‐p70 S6K (Thr389), p70 S6K (Cell Signaling Technology), phospho‐BAD (Ser136) (GeneTex, Irvine, CA, USA), BAD (Abcam, Cambridge, MA, USA), and β‐actin (Sigma‐Aldrich). Band quantification was performed using imagej^® software (National Institutes of Health, USA).

Immunoblot analyses were performed using the following antibodies: Akt, Aktp^S473, p70S6K, phospho-p70S6K, 4EBP1, ERK1/2, Atg5, ULK, ERK1/2, phopho-ERK1/2, p38, phospho-p38, phospho-NFκB and phospho-4EBP1 were all purchased from Cell Signaling. FAK (Santa Cruz Biotechnology, Inc.), actin (Cytoskeleton), LC3 (Novus) and FAKp^Y397 (BD Transduction Laboratories) were purchased from the suppliers indicated. Monoclonal anti-HA (16B12) and polyclonal anti-HA (HA.11) were both purchased from Covance. For immunofluorescence studies, LC3 (Millipore), mTOR (Cell Signaling), LAMP-1 and LAMP-2 (Developmental Studies Hybridoma Bank, University of Iowa), p62 (abcam) and ubiquitin (FK2, Enzo Life Sciences) were from the suppliers indicated. Secondary antibodies included goat anti-mouse-Cy3, donkey anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 and were purchased from Invitrogen. For flow cytometry, TLR4 (Sa15-21; Akashi et al., 2003) was conjugated to biotin and was a kind gift from Jonathan Kagan (Harvard Medical School, Boston, MA). Anti- strepavidin-APC was purchased from Biolegend.