Anti mycn sc 53993

The in-situ proximity ligation assay (isPLA) was performed as described previously elsewhere [37 (link),80 (link)]. In brief, the MYCN-amplified KELLY cell line was used to detect endogenous MYCN:MAX interactions. KELLY cells were seeded in 96-well plates for 48 h, then treated with 5 µM of MYCMI-7 or DMSO for 5 h, and then washed twice with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized with PBS with 0.05% Triton-X and incubated in a blocking buffer for 1 h at 37 °C, after which the isPLA was performed using the NaveniFlex MR kit following the manufacturer protocol (Navinci Diagnostics, Uppsala, Sweden). The following antibodies were used: mouse monoclonal anti-MYCN (sc-53993, Santa Cruz, CA, USA) and rabbit polyclonal anti-MAX (Abcam, catalog no. ab101271). Cells were stained with phalloidin and DAPI, and the isPLA signals were developed using Buffer C provided by the isPLA kit with Atto 647N. Image acquisition was performed using the ZEISS LSM 980 with an Airyscan 2 confocal microscope (Carl Zeiss Microscopy GmbH, Munich, Germany). All image stacks were acquired with comparable settings, at a resolution of 1024 × 1024 pixels and a z-stack size of 3 μm. The isPLA signals were quantified using CellProfiler 4.2.1 software. Data were analyzed using Rstudio version 4.1.0 with the following packages: tidyverse and ggplot2.