P p70s6k

Cells were lysed using RIPA lysis buffer (Solarbio) containing protease inhibitors (Beyotime) and a phosphorylase inhibitor cocktail (Roche) to obtain protein, and the contents were 990 μl RIPA lysis buffer + 10 μl PMSF + 100 μl phosphorylase inhibitor. A BCA Protein Assay Kit was used to confirm the concentrations of protein separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% BSA blocking reagent for 1 h at room temperature (RT) and incubated with primary antibodies overnight at 4° C. The membranes were washed and incubated for 1 h at RT with secondary antibodies. The proteins were analyzed using the ECL Plus kit. The following antibodies used included at a dilution of 1:1000: GABARAP (Abcam, ab109364), E-cadherin (Abcam, ab40772), N-cadherin (Abcam, ab76011), vimentin (Proteintech, 10366-1-AP), MMP2 (Abcam, ab110186), MMP14 (Abcam, ab3644), AKT (Abcam, ab179463), p-AKT (Bioworld Technology, Ser473, BS4007), mTOR (Abcam, ab2732), p-mTOR (Ser2448; 5536), p70S6K (Proteintech, 14485-1-AP), and p-p70S6K (Thr389; 9234), MEK1/2 (8727), p-MEK1/2 (Ser217/221; 9154), ERK1/2 (4695), p-ERK1/2 (Thr202/Tyr204: 4370), IKK-β (8943), p-IKK-β (2078), IκBα (4812), and p-IκBα (2859) from Cell Signaling Technology. β-actin was used as an internal control (ZSGB-BIO, TA-09, dilution 1:1500).

AML cells were cultured in complete medium containing 10% serum, stimulated with 25 ng/ml IL6 for 30 min. Cells were harvested, washed with 1 × PBS and lysated with 1 × RIPA lysis buffer containing protease/phosphatase inhibitors (Thermo Fisher #78,441). The lysates were sonicated and centrifuged at 14,000 g for 15min. Western blot analysis was performed with the following primary antibodies, including anti-4EBP1 (CST#9644S), p-4EBP1 (CST#2855P), p-mTOR (CST#5536S), mTOR (CST#2983S), p-STAT3 (CST#9145S), STAT3 (CST#4904S), p-P70S6K (CST#9205S), P70S6K (CST#35,708), CDK6 (Proteintech#14,052), CCND1 (Abcam#ab54503), GAPDH (CST#5174), and HRP conjugated secondary antibodies (Abcam#ab205718, Abcam#ab205719). Detection was conducted using a chemiluminescence substrate (Omics Bio), and images were acquired using ImageQuantTM LAS 4,000 camera and quantified using lmageJ software version1.53 (NIH, Bethesda, MD, USA). To examine whether the phosphorylated protein levels of mTOR and its downstream effectors (P70S6K and 4EBP1) were significantly inhibited, we normalized the levels of phosphorylated protein to the corresponding total protein. Uncropped images for the blots were provided in Additional file 12.

Cells were washed twice with PBS and lysed on ice with RIPA lysis buffer containing a protease inhibitor and phosphatase inhibitor. The cell lysate was centrifuged, the supernatant was collected, and the protein concentration was quantified using a BCA protein detection kit. Equal amounts of protein were used for SDS-PAGE. After separation, proteins were transferred onto a PVDF membrane and incubated with a specific primary antibody at 4 °C overnight. Then, the membrane was incubated with an HRP-conjugated secondary antibody for 1-2 hours. The immunoreaction signals were detected with a chemiluminescence reagent (Millipore, WBKLS0050) and analyzed with Image Lab software. The primary antibodies used in this experiment were specific for the following proteins: HA (Proteintech, 51064-2-AP, 1:5000), Flag (Proteintech, 66008-4-Ig, 1:5000), GST (Proteintech, 10000-0-AP, 1:5000), Ub (Proteintech, 10201-2-AP, 1:1000), HMGCL (Proteintech, 16898-1-AP, 1:1000), NEDD4 (Proteintech, 21698-1-AP, 1:1000), IKKβ (Proteintech, 15649-1-AP, 1:1000), Tubulin (Proteintech, 11224-1-AP, 1:1000), p-IKKβ (Cell Signaling Technology, #2697, 1:1000), P70S6K (Proteintech, 14485-1-AP, 1:1000), and p-P70S6K (p-Thr389) (Proteintech,28735-1-AP, 1:1000).