Rabbit mab igg

To analyze the levels of total 4E-BP1 or phosphorylated S6RP and 4E-BP1, unperturbed cells were prepared using same fixation, permeabilization and blocking protocol as for protein synthesis assays. The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA. All antibodies were used at the concentration recommended by supplier. For analysis of total 4E-BP1 levels, cells were washed and treated with 2 µg/ml secondary antibody (Goat anti-Rabbit IgG secondary antibody conjugated to Alexa Fluor 568, #A-11011, Thermo Fisher Scientific) for 2 hr in RT. Antibodies were washed away using 5% BSA in PBS and the cells were stained for p-Histone H3 (S10) and DNA, as in protein synthesis assays. Finally, the cells were washed three times with PBS, mixed in to PBS supplemented with 1% BSA and put on ice until FACS analysis.

Antibodies against USP4 (#2651), ERK1/2 (#4695), phospho-ERK1/2 (#4370), CrkII (#3492), phospho-CrkII (#3491), PARP (#9532), cleaved-PARP (#5625), cleaved-caspase3 (#9661), HA-tag (#3724), Myc-tag (#2276), Flag-tag (#14793), rabbit mAb IgG (#3423), and mouse mAb IgG (#3420) were purchased from Cell Signaling Technology (USA). Antibodies targeting CypA (ab41684, ab58144), MMP-9 (ab38898), and Ki-67 (ab15580) were purchased from Abcam (UK). Antibodies against USP4 (for Co-IP and IHC, sc-376000), p38 (sc-7972), p-p38 (sc-7973), JNK (sc-7345), P-JNK (sc-6254) were purchased from Santa Cruz Biotechnology (USA). Anti-β-actin (AF0003), anti-GAPDH (AF0006), anti-mouse IgG (HRP) (A0216), and anti-rabbit IgG (HRP) (A0208) antibodies were purchased from Beyotime Biotechnology (China).

Antibodies for immunoblotting such as p‐AMPKα (#2535), AMPKα (#5831), PDK1 (#3820), HIF1α (#3716), hexokinase 2 (#2867), LDHA (#3582), PFKP (#8164), PGAM1 (#12098), PDH (#3205), p‐Threonine/Tyrosine (#9381), p‐S6K (#9234), S6K (#2708), p‐ERK (#4370), ERK (#4695), p‐4E‐BP1 (Thr37/46, #2855), p‐4E‐BP1 (Ser65, #9451), 4E‐BP1 (#9644), mouse antirabbit IgG mAb (#5127), and antirabbit IgG (#7074) were from Cell Signaling Technology (Danvers, MA, USA). HIF1α (hydroxyl P564) (ab72777) and PHD3 (ab30782) were purchased from Abcam (Cambridge, MA, USA). HIF1α (hydroxyl P402) (07‐1585) was purchased from Merck (Darmstadt, Germany). GAPDH (sc‐25778) was purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). Actin (GTX109639) was purchased from GeneTex International (Hsinchu, Taiwan). Antibodies for immunoprecipitation such as HIF1α (#36169), rabbit mAb IgG (#3900), and p‐AMPKα (#2535) were purchased from Cell Signaling Technology. AMPKα1 (sc‐398861) was purchased from Santa Cruz Biotechnology. Antibodies for IHC such as p‐AMPKα (GTX52341) and HIF1α (GTX127309) were purchased from GeneTex International.