Phospho akt

Antibodies used for this study were directed against phospho-BCR-ABL (210 kDa, 3901S), H3K27me3 (17 kDa, 9733), PARP (89 & 116 kDa), Akt (60 kDa, 9272S), KDM6A (155 kDa, 33510) from CST (Danvers, MA, USA); BCR-ABL (210 kDa, ab187831), TRKA (145 kDa, ab8871), H3K27ac (17 kDa, ab4729) from abcam (MA, USA); Flag (T0003), YY1 (70 kDa, 22156-1-AP), beta actin (42 kDa, 20536-1-AP), c-FOS (60 kDa, 66590-1-lg), CBP (29 kDa, 11149-1-AP) from Proteintech, IL, USA; KDM6A (155 kDa, A302-374A, Bethyl Laboratories, Montgomery, Texas, USA); phospho-AKT (60 kDa, GTX128414, GeneTex, CA, USA); phospho-Erk (44 kDa, 11245), Erk (42/44 kDa, 44204) from SAB, Maryland, USA; GAPDH (36 kDa, AP0063, Bioworld, Nanjing, China); RNA pol II (217 kDa, 39497) from Active Motif, Carlsbad, CA.

Cell lysates were separated by SDS/PAGE (8, 10 or 12% polyacrylamide gel), electrotransfered to PVDF membrane (Millipore,) and blocked for 1 h (at room temperature) in 5% non-fat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (BioShop, Burlington, Canada). Membranes were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody for 1 h, room temperature, chemiluminescence was detected using Immobilon Western HRP substrate (Millipore) with ChemiDoc system (BioRad). The following antibodies and dilutions were used: rabbit anti-MCPIP1 (1:1000, GeneTex), HIF1α (1:1000), HIF2α (1:1000), VHL (1:500), Akt (1:1000), Phospho-Akt (1:2000), SAPK/JNK (1:500), Phospho-SAPK/JNK (1:1000), p38 (1:1000), Phospho-p38 (1:1000), ERK1/2 (1:1000), Phospho-ERK1/2 (1:1000). All mentioned antibodies were from Cell Signaling Technology. Tubulin (1:4000, Calbiochem; Merck Millipore, Billerica, MA, USA) or in case tissue samples, GAPDH (1:30,000 Sigma-Aldrich) antibodies were used as a loading control. The following secondary antibodies were used: peroxidase-conjugated anti-rabbit IgG (1:30,000; Sigma) and peroxidase-conjugated anti-mouse IgG (1:10,000, Sigma).

The immunoblotting assay was executed as previously described [29 (link)]. Briefly, the proteins were separated with 8–12% SDS-PAGE and were transferred onto polyvinylidene fluoride membranes (PVDF). The membrane was then incubated with the primary antibodies of Akt, phospho-Akt, ZO-1 (cell signaling), E-cadherin, N-cadherin, vimentin, and GAPDH (GeneTex, Inc., CA, USA) for 1 h at room temperature or overnight at 4 °C. After the HRP secondary antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) administration, the signals were detected using a Trident femto Western HRP Substrate (GeneTex) and exposed to a ChemiDoc^TM XRS Imaging System (BIO-RAD) for autoradiography.