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. 2019 Apr 7;11(4):492. doi: 10.3390/cancers11040492

Figure 2.

Figure 2

ANXA2 via its Cys-8 REDOX sensitive residue binds to PTEN and regulates its activity. (a) 293T cells transfected with either empty vector; ANXA2 and PTEN-GFP expression plasmids or ANXA2 Cys-8-Ala and PTEN-GFP expression plasmids were grown in 60 mm plates. Cells were washed with PBS, lysed with Triton X-100 lysis buffer degassed for 1 h on ice. 300 µg of each cell lysate was incubated with the indicated antibodies for 1 h at 4 °C and then 50 µL of pre-washed 50% slurry protein G-Sepharose™ (GE Healthcare) was added for 1 h at 4 °C. Beads were washed four times with 300 μL of lysis buffer, resuspended with 20 μL 2× SDS-PAGE loading buffer, boiled for 10 min, subjected to SDS-PAGE and analyzed by western blotting; 293T cells over-expressing (b) (1) empty vector, (2) PTEN, (3) ANXA2 or (4) ANXA2 and PTEN; or (c) (1) ANXA2 and PTEN, (2) ANXA2 Cys-8-Ala and PTEN, (3) ANXA2 Cys-132-Ala and PTEN, (4) ANXA2 Cys-334-Ala and PTEN, (5) ANXA2 and PTEN Cys-124-Ser or (6) pcDNA3 (empty vector) were grown in 60 mm plates in DMEM complete medium for 24 h. Cell lysates were prepared and 20 µg of each protein extract was subjected to SDS-PAGE, transferred onto nitrocellulose membranes and analyzed by western blotting with the antibodies indicated.