Figure 1. PIPKI-α is required for... | Rockefeller University Press

Figure 1.

$Figure 1. PIPKI-α is required for adhesion-induced Rac1 plasma membrane targeting. (A) HeLa cells were incubated for 20 min with 5 µm FN- or BSA-coated beads shown in the phase-contrast image (differential interference contrast [DIC]) and marked by arrows. Cells were fixed and stained with Alexa Fluor 647 phalloidin to visualize F-actin or with appropriate primary and secondary antibodies to detect endogenous PIPKI-α and Rac1. (B) HeLa cells, transiently transfected with GFP–PLC-δ–PH as a marker for PI4,5P2, were incubated with FN- or BSA-coated beads as described above, and GFP fluorescence and DIC images were recorded. (C) Cells transfected with control siRNAs or siRNAs targeting PIPKI-α, -β, -γ, or Rac1 were incubated with FN-coated beads, fixed, and stained as described in A. (right) Fluorescence intensity profiles showing the extent of marker colocalization were determined along the lines. Colors indicate PIPKI-α (red), Rac1 (green), and F-actin (blue) fluorescence. Images in A–C are representative examples of three separate experiments. (D) Subcellular fractionation of HeLa cell lysates into particulate (P) and soluble (S) fractions. HeLa cells transfected with the indicated siRNA pools were plated on FN-coated substrate and serum starved to eliminate contribution from growth factor signals. Cell lysates were generated, subfractionated into 100,000 g pellet (P100; membrane) and supernatant (S100; cytosol) fractions, and analyzed by Western blotting with antibodies against PIPKI-α and Rac1. EGFR and RhoGDI were detected as markers for membrane and cytosol fractions, respectively. A representative experiment is shown. Note that the amount of protein analyzed per lane represents 5% of the total soluble fraction but 20–50% of the particulate fraction. (E) Quantification of Rac1 membrane localization by quantitative Western blot (WB) analysis from D is shown. Data are normalized according to the total levels of each marker and the calculated percentage in the membrane fraction. Values are means from two independent experiments. Error bars indicate SEM. Bars, 10 µm.$

PIPKI-α is required for adhesion-induced Rac1 plasma membrane targeting. (A) HeLa cells were incubated for 20 min with 5 µm FN- or BSA-coated beads shown in the phase-contrast image (differential interference contrast [DIC]) and marked by arrows. Cells were fixed and stained with Alexa Fluor 647 phalloidin to visualize F-actin or with appropriate primary and secondary antibodies to detect endogenous PIPKI-α and Rac1. (B) HeLa cells, transiently transfected with GFP–PLC-δ–PH as a marker for PI4,5P₂, were incubated with FN- or BSA-coated beads as described above, and GFP fluorescence and DIC images were recorded. (C) Cells transfected with control siRNAs or siRNAs targeting PIPKI-α, -β, -γ, or Rac1 were incubated with FN-coated beads, fixed, and stained as described in A. (right) Fluorescence intensity profiles showing the extent of marker colocalization were determined along the lines. Colors indicate PIPKI-α (red), Rac1 (green), and F-actin (blue) fluorescence. Images in A–C are representative examples of three separate experiments. (D) Subcellular fractionation of HeLa cell lysates into particulate (P) and soluble (S) fractions. HeLa cells transfected with the indicated siRNA pools were plated on FN-coated substrate and serum starved to eliminate contribution from growth factor signals. Cell lysates were generated, subfractionated into 100,000 g pellet (P100; membrane) and supernatant (S100; cytosol) fractions, and analyzed by Western blotting with antibodies against PIPKI-α and Rac1. EGFR and RhoGDI were detected as markers for membrane and cytosol fractions, respectively. A representative experiment is shown. Note that the amount of protein analyzed per lane represents 5% of the total soluble fraction but 20–50% of the particulate fraction. (E) Quantification of Rac1 membrane localization by quantitative Western blot (WB) analysis from D is shown. Data are normalized according to the total levels of each marker and the calculated percentage in the membrane fraction. Values are means from two independent experiments. Error bars indicate SEM. Bars, 10 µm.