Hc-TeTx and nSM2 show a similar distribution in the plasma membrane.

<p><b>A)</b> CGN cultures were incubated with Hc-Alexa 488 (10 nM) for 15 min and subsequently fixed and labeled with the nSM2 antibody. Merge image show that both proteins have a punctuated pattern and partial colocalization, as is highlighted by the magnification. Nuclei are stained with DAPI (blue). <b>B)</b> Extraction of detergent-resistant membranes (DRM) from CGN with 1% Brij 98 shows that nSM2 partially associates with membrane rafts, as has been described for TeTx and for Hc. Transferrin receptor (TfR) is used as marker of soluble membrane while Flotillin-1 is used as marker of DRM. <b>C)</b> CGN were treated with MCD 5 mM for 30 min, treatment that extracts cholesterol from the membranes and turns DRM sensitive the detergent treatment, as can be seen thanks to the signal increase of both Flotillin-1 and nSM2 in the soluble fractions (SF) respect to the control condition (<i>C</i>). Hc treatment (10 nM for 30 min) does not cause any change in nSM2 association to DRM. <b>D)</b> CGN cultures were incubated with Hc-Alexa 488 as in A and subsequently fixed and labeled with the anti-ceramide antibody. Merge image show that both proteins have a punctuated pattern and a partial colocalization, as is highlighted in the magnification. Nuclei are stained with DAPI (blue).