Technical Reference #1613

1613.

Subcellular Localization and Lysophospholipase/ Transacylation Activities of Human Group IVC Phospholipase A2 (cPLA2gamma) Atsushi Yamashita ⁎; Ken Tanaka; Ryo Kamata; Tsukasa Kumazawa; Naotaka Suzuki; Hiroki Koga; Keizo Waku; Takayuki Sugiura, Teikyo University, Biochimica et Biophysica Acta, 1791(1613), (2009)
Link To Paper

Abstract:
cPLA2γ was identified as an ortholog of cPLA2α which is a key enzyme in eicosanoid production. cPLA2γwas reported to be located in endoplasmic reticulum (ER) and mitochondria and to have lysophospholipaseactivity beside phospholipase A2 (PLA2) activi

Keywords:
CAAX Box Motif; lysophospholipid; phospholipase A2; cPLA2gamma; lysophospholipase; lysophospholiase; transacylation

Materials & Methods:
2.4. Immunofluorescence microscopy HeLa cells were plated on 35 mm glass-bottomed MatTek plates at 1.25×104 cells/cm2 and transfected with the expression plasmids as described above. The cells were rinsed once with PBS and fixed for 15 min in ice-cold 4% paraformaldehyde in PBS before rinsing with cold PBS and permeabilization by incubation for 15 min in 1% Triton X- 100 in PBS. Permeabilized cells were incubated with anti-FLAG mouse monoclonal antibody (2.5 μg/ml) for 60 min followed by 45 min incubation with anti-mouse IgG AlexaFluor 488-conjugated secondary antibody (1:100). Cells were counterstained with anti-calnexin polyclonal antibody (1:100) and an AlexaFluor 546-conjugated anti-rabbit secondary antibody (1:100). To counterstain with anti-Tom20 (mitochondria) or anti-GM-130 (Golgi) mouse monoclonal antibodies anti- FLAG rabbit polyclonal antibody was used for cPLA2γ staining. Stained cells were visualized using a Leica TCS-SP5-DIM6000 confocal laser microscope system (Leica Microsystems).

Microscopic Technique
Fluorescence Microscopy, Immunofluorescence

Cell Type(s)
HeLa, HEK293