Anti phospho p70s6k thr389

Cultured naïve CD4⁺ T cells were lysed with RIPA buffer (Sigma–Aldrich) with protease inhibitor cocktail (Roche Diagnostics). Primary antibodies used for immunoblotting were as follows: anti-Akt, anti-phospho-Akt (Thr308), anti-Stat3, anti-phospho-Stat3 (Tyr705), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-p70S6K, anti-phospho-p70S6K (Thr389), anti-S6, anti-phospho-S6 (Ser240/244) and anti-β-actin were purchased from Cell Signalling Technology. Anti-Bcl6 (clone: IG191E/A8) were obtained from Biolegend. Followed by horseradish peroxidase-conjugated (HRP) conjugated secondary antibodies (Anti-rabbit IgG), and detected by chemiluminescent substrates (GE Healthcare Biosciences). Gel bands were analysed with Photoshop CS4 software (Adobe), and fold changes were analysed with ImageJ software (NIH).

Cell lysates (20 μg protein each) were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transblotted onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk and incubated with anti-phospho-p70^S6K (Thr389) (1:1,000) (Cell Signaling), anti-Twist (1:1,000) (Santa Cruz), anti-Sox9 (1:1,000) (Abcam), anti-N-cadherin (1:1,000) (Zymed), anti-E-cadherin (1:1,000) (BD Transduction Laboratories) in phosphate-buffered saline containing 0.1% Tween 20 rotating at 4°C overnight. After washing, the membrane was further incubated with secondary antibodies coupled to horseradish peroxidase at room temperature for 1 h and developed with an enhanced chemiluminescent detection kit (Amersham).

Tissue culture media and reagents (Dulbeco’s modified Eagle’s media, FBS, trypsin, penicillin/streptomycin, were from Sigma. PHD inhibitor IOX2 was from MedChem Express (cat. number: HY-15468). Plasmids encoding HIF1α P402A/P564A (HA-HIF1alpha P402A/P564A-pcDNA3) and HIF2α P405A/P531A (HA-HIF2alpha-P405A/P531A-pcDNA3) were from Addgene (cat. numbers: 18955 and 18956). CD63-GFP (CD63-pEGFP C2) and GFP (mEGFP-C1) were from Addgene (cat. numbers: 62964, and 54759). pKLpuro plasmids expressing validated shRNAs for HIF1α and HIF2α were obtained from Sigma-Merck (MISSION shRNAs library) and expressed as a pool of 4–5 shRNAs per each target gene Antibodies: anti-HIF1α was from BD Biosciences, (cat. number: 610958) or Cayman (cat. number: 000 6421) and used at 1:1000, anti-HIF2α was from Bethyl, UK (cat. number: A700-003) used at 1:2,000 or Santa Cruz (cat. number: Sc-13596) used at 1:500; anti-β-tubulin was from Merck-Sigma Aldrich, (cat. number: T4026) and used at 1:2000; anti-phospho-P70S6K(Thr389) was from Cell Signalling (cat. number: #9234) and used at 1:1000; anti-p62/SQSTM1 was from Progen (cat. number: GP62-C) and used at 1:1000; anti-phospho-eIF2α was from Abcam (cat. number: Ab32157) and used at 1:1000. For immunofluorescence studies antibodies against HIF1α and HIF2α were from Cayman and Santa Cruz respectively, (Cat. Numbers: 10006421 and Sc-13596) and used at 1:100).

The following primary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): anti-BiP (#3177), anti-LC3A/B (#4108), anti-Phospho-p70S6K (Thr389) (#9205), anti-p70S6K (#9202), anti-Phospho-ULK1 (Ser757) (#14202), anti-ULK1 (#6439), anti-phospho-PERK (Thr980) (#3179), anti-PERK (#3192), anti-Phospho-eIF2-alpha (Ser51) (#9721), anti-eIF2-alpha (#9722) anti-Tuberin/TSC2 (#4308). Anti-β-actin and anti-α-tubulin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-p62 (GP62-C-WBC) was obtained from ProGen (Heidelberg, Germany). Anti-Lamin A/C/B1 (EPR4068) was obtained from Abcam. Anti-prelamin A (#MABT345) was obtained from Millipore. Anti-p27 Kip1/CDKN1B (sc-1641) was obtained from Santa Cruz Biotechnology. The following secondary antibodies HRP-conjugated were used: anti-Rabbit (NA934) and anti-Mouse (NA931) were obtained from GE Lifesciences (Marlborough, MA, USA). Anti-Guinea pig HRP-conjugated (90001) was purchased by ProGen (Heidelberg, Germany). Chloroquine (C6628) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cells were lysed in ice-cold lysis buffer (20 mMTris-HCl (pH 7.5), 150 mM NaCl, 1%Triton-X 100, 1 mM EDTA and a protein inhibitor cocktail) for 30 min. The supernatant was boiled with Laemmli sample buffer for SDS-PAGE. Antibodies as follows: anti-Rheb and anti-FADD from Abcam, anti-LC3B, anti-SQSTM1/p62, anti-p70s6k, anti-phospho-p70s6k (Thr389) and anti-ATG5 from Cell Signaling Tech, and other antibodies: anti-α-Tubulin (Epitomics, 2871-1), anti-Flag (Sigma-Aldrich, F7425), anti-GAPDH (Santa Cruz Biotechnology, L-3113). Band intensity was quantified by ChemiAnalysi software (Bioshine, China).

The following antibodies were used in the experiments: anti-Flag (F3165, 1:2000) from Sigma-Aldrich; anti-GFP (11814460001, 1:2000), anti-Myc (11667149001, 1:2000) and anti-HA (11583816001, 1:2000) from Roche Applied Science; anti-CUL2 (ab166917, 1:500) and anti-GLUT1 (ab115730, 1:1000) antibodies from Abcam. Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti-­mouse IgG horseradish peroxidase (HRP)-­conjugated whole antibody (31430, 1:50,000) and goat anti-­rabbit IgG-HRP-­conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific. Cy3-labeled goat anti-rabbit or mouse IgG was purchased from CWBio.

Western blot analysis was performed on tissue homogenates as described [13 (link)]. The antibodies used were anti-phospho-AKT (Ser473), anti-phospho-p44/p42-MAPK (Thr202/Tyr204) and anti-phospho-p70S6K (Thr389) from Cell Signaling Technology (Danvers, MA, USA); anti-IRβ from Santa Cruz Biotechnology (Dallas, TX, USA) and anti-β-actin from Sigma-Aldrich Corp (St. Louis, MO, USA). Fifty micrograms of whole proteins were separated on an 8–15% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Immobilon P; Millipore, Burlington, MA, USA). The membrane was blocked in washing solution with 5% non-fat dried milk for 60 min at 37 °C and then incubated with 1 μg/mL primary antibody overnight at 4 °C. The primary antibodies were immunodetected using a horseradish peroxidase-conjugated secondary antibody for 60 min at 37 °C. The bands were detected with a chemiluminescent system (ECL; GE Healthcare, Piscataway, NJ, USA) and exposed to X-ray film. The band intensities were quantified using ImageJ v1.52k software (http://rsb.info.nih.gov/ij, accessed on 20 January 2021).