Phospho 4e bp1t70

HA-tagged and FLAG-tagged eIF4E expression plasmids were constructed by cloning eIF4E to AfeI and SbfI sites on the pLVX-EF-puro plasmid (53 (link)). pLVX-EF-4E-BP1^WT and pLVX-EF-4E-BP1^S83A expression plasmids were generated based on previous constructs (34 (link)). Doxycycline-inducible 4E-BP1^T37A and 4E-BP1^T46A plasmids were constructed by cloning corresponding 4E-BP1 mutant fragments to AfeI and SbfI sites on the pLenti-TRE-MCS-EF-Puro-2A-rTet plasmid (54 (link)). DNA constructs used in this study are listed in Table S1.
The following primary antibodies were used in this study: total 4E-BP1 (53H11, Cell Signaling Technology), phospho-4E-BP1^T37/T46 (236B4, Cell Signaling Technology), phospho-4E-BP1^T70 (9455, Cell Signaling Technology), phospho-4E-BP1^S65 (9451, Cell Signaling Technology), phospho-4E-BP1^S83 (ABE2889, Millipore), eIF4E (C46H6, Cell Signaling Technology), eIF4GI (D6A6, Cell Signaling Technology), eIF4E (A-10, Santa Cruz Biotechnology), eEF2 (2332, Cell Signaling Technology), HA tag (16B12, BioLegend), and FLAG tag (M2, Sigma-Aldrich).

Samples containing equal amounts of protein (10 μg) were resolved by polyacrylamide gel electrophoresis (SDS-PAGE) and processed as described previously [15 (link),16 (link)]. Briefly, membranes were blocked using TBS-Tween containing 3% (w/v) BSA for 1 hour and incubated with antisera diluted in the same overnight at 4°C. Following washing in TBS-Tween, membranes were incubated with horseradish peroxidase-conjugated secondary antibody and signals developed using ECL reagent. The antiserum used were raised against: eIF4A, phospho-eIF4E, eIF4G, eIF4E [15 (link),16 (link)]; MGMT, phospho-eIF2α, (Abcam, UK); 4E-BP1, phospho-4E-BP S65, phospho-4E-BP1 T70, phospho-p70 S6K T389, phospho- ERK1/2, phospho-p38 MAPK, phospho-rpS6 S240/44, phospho-Akt T308, p21^cip1 (Cell Signaling, UK).

Cycloheximide, 5-aza-2′-deoxycytidine (5-Aza), trichostatin A (TSA), and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The indicated primary antibodies against the following proteins were used in this study: STAT1/2/3/4/5/6 (#9172/4594/12640/5097/9363/9362), DNMT1/3a/3b (#5119/3598/67259), cleaved-caspase 3/7 (#9664/9491), cleaved PARP (#5625), S6K (#2708), phospho-S6K (T389) (#9205), S6 (#2217), phospho-S6 (Ser235/236) (#2211), mTOR (#2972), 4E-BP1 (#9644), phospho-4E-BP1 (T70) (#9455), eIF4E (#2067), phospho-eIF4E (S209) (#9741), eIF4G (#2469), Rheb (#13879), TSC2 (#4308), p-TSC2 (T1462) (#3617), p-TSC2 (S1387) (#5584), ICAM1 (#4915), JAK2 (#3230), NFATC2 (#4389) and Myc taq (#2278) (Cell Signaling Technology, Danvers, MA, USA); anti-HIF-1α (610958) (BD Biosciences, San Jose, CA, USA); and anti-actin (sc-1616) and GAPDH (sc-48,167) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies used were anti-goat IgG HRP (81–1620; Invitrogen, Carlsbad, CA, USA), anti-mouse IgG HRP (G-21040; Invitrogen), and anti-rabbit IgG HRP (111–035-003; Jackson Laboratories, Bar Harbor, ME, USA).