1. Gα12 and Gα13 Interact with Ser/Thr Protein Phosphatase Type 5 and Stimulate Its Phosphatase Activity 
Yoshiaki Yamaguchi, Hironori Katoh, Kazutoshi Mori, Manabu Negishi 
Current Biology 
Volume 12, Issue 15, Pages (August 2002)
DOI: /S (02)

2. Figure 1 In Vitro Interaction of Gα12QL with PP5
(A) Diagrams of full-length PP5, GST-fused PP5-TPR, PP5-PD, and PP5. The numbers of corresponding amino acids are also shown.
(B) COS-7 cells were transiently transfected with an expression vector encoding Gα12QL, Gα13QL, GαqQL, or Gαi2QL, and cell lysates were incubated with GST (lane 2) or GST-fused PP5-TPR (lane 3). Cell lysates expressing Gα12QL were also incubated with GST-fused PP5-PD (lane 5) or GST-fused PP5 (lane 6). They were then immobilized on glutathione-Sepharose beads, and bound G proteins and lysate input (lysate, lanes 1 and 4) were analyzed by immunoblotting with anti-Gα12, Gα13, Gαq, or Gαi2 polyclonal antibody. The results shown are representative of three independent experiments that yielded similar results.
(C) Purified bacterially expressed GST and GST-fused PP5-TPR, PP5-PD, and PP5 used in this experiment (arrowheads). Approximately 2 μg of each protein was electrophoresed in a 12.5% SDS polyacrylamide gel and stained with Coomassie brilliant blue. Molecular mass markers are shown on the left.
Current Biology  , DOI: ( /S (02) )

3. Figure 2 In Vivo Interaction of Gα12 with PP5
(A) COS-7 cells were transiently cotransfected with expression vectors encoding Myc-tagged PP5 and either Gα12WT or Gα12QL; alternatively, they were transiently cotransfected with expression vectors encoding Myc-tagged PP5-PD and Gα12QL. Cell lysates were then immunoprecipitated with anti-Gα12 polyclonal antibody in the absence or presence of AlF4− as indicated. G proteins in immunoprecipitates (right, upper panel) and lysate input (left, upper panel) as well as Myc-tagged PP5 (arrow) and PP5-PD (arrowhead) in immunoprecipitates (right, lower panel) and lysate input (left, lower panel) were analyzed by immunoblotting with anti-Gα12 polyclonal antibody and anti-Myc monoclonal antibody, respectively.
(B and C) About 10 mg of adult rat brain lysate was immunoprecipitated with either anti-Gα12, anti-Gαq, or anti-PP5 antibody and immunoblotted with these antibodies. Expressions of Gα12, Gαq, and PP5 in adult rat brain lysate are also shown. The results shown are representative of three independent experiments that yielded similar results.
Current Biology  , DOI: ( /S (02) )

4. Figure 3 Subcellular Localization of PP5 in COS-7 Cells
(A) Immunofluorescence confocal microscopy. COS-7 cells were transiently transfected with an expression vector encoding Myc-tagged PP5 along with an empty vector (panel a) or a vector encoding either Gα12QL (panels b and c) or Gαi2QL (panels f and g); alternatively, they were transfected with an expression vector encoding Myc-tagged PP5-PD along with a vector encoding Gα12QL (panels d and e). At 8 hr after transfection, cells were fixed and stained with anti-Myc monoclonal antibody (panels a, c, e, and g) and with either anti-Gα12 polyclonal antibody (panels b and d) or anti-Gαi2 polyclonal antibody (panel f). The bar represents 10 μm.
(B) Immunofluorescence microscopy. COS-7 cells transfected with Myc-tagged PP5 and Gα12QL were stained as described above. The nucleus was also stained with DAPI. The bar represents 10 μm.
(C) Biochemical fractionation. Cellular homogenates from COS-7 cells transfected with a vector encoding Myc-tagged PP5 along with an empty vector (lanes 1 and 2) or with a vector encoding Gα12QL (lanes 3 and 4) were separated into cytosolic (C, lanes 1 and 3) and membrane (M, lanes 2 and 4) fractions. The fractionated samples were analyzed by immunoblotting with anti-PP5 monoclonal antibody and anti-Gα12 polyclonal antibody. The results shown are representative of three independent experiments that yielded similar results.
Current Biology  , DOI: ( /S (02) )

5. Figure 4 Effect of Gα12QL on PP5 Phosphatase Activity In Vitro
(A) Activation of GST-PP5 by GST-Gα12QL, GST-Gα13QL, and activated GST-Gα12WT. GST-PP5 phosphatase activity was measured in the absence or presence of 10 nM GST or GST-fused GTPases. Where indicated, 100 μM of GDPβS or GTPγS was added.
(B) Synergistic effect of GST-Gα12QL and arachidonic acid on GST-PP5 phosphatase activity. GST-PP5 phosphatase activity was measured at the indicated concentrations of arachidonic acid in the absence or presence of 10 nM GST or GST-fused GTPases. Symbols: open diamonds, none; open squares, GST; open triangles, GST-GαqQL; open circles, GST-Gαi2QL; filled triangles, GST-Gα12QL; and filled circles, GST-Gα13QL.
(C) GST-Gα12QL had no effect on GST-PP5-PD phosphatase activity. GST-PP5 or GST-PP5-PD phosphatase activity was measured under the indicated combinations. The results are the means ± S.E. of triplicate experiments.
Current Biology  , DOI: ( /S (02) )