Flow Cytometry analysis of HepG2 cells using anti-GGA2 antibody. Overlay histogram showing HepG2 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-GGA2 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Western blot analysis of GGA2 using anti-GGA2 antibody. Lane 1: human THP-1 whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: human K562 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-GGA2 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. A band at ~70 kDa is detected across cell lysates, appearing as a tight doublet in some samples. The small upward shift from the ~67 kDa prediction and the doublet pattern are consistent with phosphorylation-dependent mobility differences reported for GGA2.

Immunoprecipitating GGA2 in HepG2 whole cell lysate. Western blot analysis of GGA2 using anti-GGA2 antibody; Lane 1: HepG2 whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-GGA2 antibody in HepG2 whole cell lysate; Lane 3: anti-GGA2 antibody (2ug) + HepG2 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-GGA2 antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. The expected molecular weight of GGA2 is at 67 kDa.