Protein fractionation kit

Cells were collected and processed for protein isolation using T-PER, (tissue protein extraction reagent) for tissue and Pierce RIPA buffer for tumor cells (Thermo Scientific, USA). Total protein was separated by Tris/Glysine/SDS gel electrophoresis. Membranes were incubated with primary antibodies against HIF1α (1:1000, R&D), VEGFR2 (1:1000, Abcam), phospho-P38, total-P38, phospho-ERK, total-ERK phospho-AKT and total-AKT (1:1000, Cell signaling), ID1 (1:1000, Biocheck, USA), β-actin (1:5000, Sigma), and HRP-conjugated secondary antibody (1:5000, Biorad).
For nuclear translocation studies, tumor cells were collected after 48 hours of cell culture and proteins from sub-cellular compartments were prepared using protein fractionation kit (Thermo Scientific, USA). VEGFR2 (Abcam) were detected in all compartments. Each of the compartments had different loading controls (Abcam): cell membrane (HSP-60), Nuclear (Lamin A/C), chromatin (Histone H3), cytoplasmic and cytoskeletal (β-actin, Sigma). Western blot images were acquired by Las-3000 imaging machine (Fuji Film, Japan).

Western blot and co-immunoprecipitation (co-IP) analysis were performed as previously described^{57 (link)}. All extracts were prepared in Laemmli buffer and boiled for 10 min. All experiments were performed at least 3 times. Sub-cellular fractionation was performed using the protein fractionation kit (78840; Thermo-Fisher) according to manufacturer’s instructions. Densitometry quantification was performed using ImageJ (version 1.49p) software by measuring the area under the curve for the band intensity in each lane. See Supplementary Table 5 for a full list of antibodies used for western blot and immunoprecipitation.