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. 2021 Jun 29;4(9):e202101095. doi: 10.26508/lsa.202101095

Figure 4. Relationships between PI(4,5)P2 and RhoB-GTP/connector enhancer of kinase suppressor of Ras1 (CNKSR1) interaction at the plasma membrane.

Figure 4.

(A) Confocal images of SKBR-3 cells treated with control siRNA, CUL3 siRNA #1 or KCTD10 siRNA #1 for 72 h. 48 h after transfection of the GFP-PLC-PH vector (a biosensor for PI[4,5]P2), cells were fixed. Bars; 10 μm. (B) Confocal images of SKBR-3 cells treated with control siRNA or KCTD10 siRNA #1 for 72 h. 48 h after infection of the Myc-CNKSR1–carrying lentivirus, cells were fixed, permeabilized, and stained for Myc and RhoB. Cells were treated with PHT-7.3 (50 μM) for 24 h before the fixation. Membrane ruffles were indicated by arrowheads. A fluorescence intensity profile along the arrow in the image is shown in right panel. PM, plasma membrane. Bars; 10 μm. (C) In vitro binding assay for determination of the biotinylated CNKSR1/FLAG-tagged proteins interaction using AlphaScreen technology. Proteins synthesized by wheat germ extracts (Fig 2E) were subjected to AlphaScreen as indicated. DHFR was used as a negative control. The red lines indicate the threshold of interactions (10 times the luminescence signal for CNKSR1/DHFR). Data from three independent experiments are expressed as the means ± SEM. The protein mixtures were incubated with PHT-7.3 for 1 h.