PIK3C3 Antibody / VPS34 / Phosphatidylinositol 3-kinase catalytic subunit type 3 (FY13075)

Flow Cytometry analysis of SH-SY5Y cells using anti-PIK3C3 antibody. Overlay histogram showing SH-SY5Y 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-PIK3C3 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 without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of VPS34/PIK3C3 using anti-PIK3C3 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela whole cell lysates, Lane 2: human 293T whole cell lysates, Lane 3: human SH-SY5Y whole cell lysates, Lane 4: human Jurkat 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-PIK3C3 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 an ECL Plus Western Blotting Substrate. A predominant band is detected at ~110-120 kDa, running above the calculated ~102 kDa, consistent with the known migration of Vps34 within class III PI3K complexes and possible phosphorylation-dependent mobility. A lighter band at ~65 kDa is also observed and likely represents a proteolytic fragment of Vps34 that can appear in stressed samples or with incomplete protease inhibition.