Figure 6.

Colocalization of SMase D and Cholera Toxin subunit b in human keratinocytes membrane. HaCaT cells cultured on slides were treated, for 2 h, with buffer or SMase D (5 µg/ml). Binding of SMase D was analyzed using rabbit IgG anti-SMase D (1:200), followed by anti-rabbit secondary antibody conjugated with Alexa-488 (1:200). Lipid rafts were visualized using Cholera toxin subunit b-AlexaFluor 555 and the nuclei counterstained with DAPI and slides were analyzed by CLSM. Scale bars represent 20 µm. (A) Cells treated with buffer or SMase D, at focal plane of 2.59 µm. Colocalization areas are shown as grayscale images. (B) average percentage SMase D/CTx-b colocalization represent means ± SEM from, at least, 10 images in two independent experiments and three different focal plans. (C) Cells treated with SMase D at focal planes from 0.86 to 5.18 µm. Colocalization areas are shown as grayscale images. (D) Percentage of SMase D/CTx-b colocalization at the different focal planes analyzed. Data on the graphs represent means ± SEM from, at least, 10 images in two independent experiments and three different focal plans.