Computational protocol: A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors

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Protocol publication

[…] Four days after transfection, the cells were lysed in ice-cold cell extraction buffer (Thermo Fisher Scientific, FNN0011) supplemented with EDTA-free protease inhibitor mixture (Sigma-Aldrich Sweden AB, 11873580001) and 1 mm phenylmethylsulfonyl fluoride. Samples were denatured with NuPAGE-LDS sample buffer (Thermo Fisher Scientific, NP0007) supplemented with NuPAGE sample-reducing agent (Thermo Fisher Scientific, NP0009) at 100 °C for 5 min, and then separated on a NuPAGE Novex 3–8% Tris acetate gel (Thermo Fisher Scientific, EA0375BOX) under reducing conditions by adding NuPAGE antioxidant (Thermo Fisher Scientific, NP0005). PON2 and RAB4A proteins were separated on a NuPAGE Novex 10% BisTris gel (Thermo Fisher Scientific, NP0302BOX). The separated proteins were transferred to polyvinylidene difluoride (Thermo Fisher Scientific, LC2002) or nitrocellulose (Bio-Rad Laboratories AB, Solna, Sweden, 170-4270) membranes using a Trans-Blot Turbo Blotting System (Bio-Rad Laboratories AB). The blot was blocked with 5 or 2% nonfat dry milk in TBST (200 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Tween 20) and then incubated with primary antibodies diluted in 2.5 or 1% nonfat dry milk in TBST overnight at 4 °C or for 45 min at room temperature. For PON2 and RAB4A, Odyssey blocking buffer was used (LI-COR Biotechnology, 927-40000), and primary antibodies were diluted in this buffer. HRP-conjugated secondary antibodies and an Amersham Biosciences ECL Select Western blotting detection reagent kit (GE Healthcare, RPN2235) were used to detect the proteins. Images were acquired and analyzed with a ChemiDoc Touch Imaging System (Bio-Rad Laboratories AB) and Image Lab software (version 5.2.1). For PON2 and RAB4A, fluorescent secondary antibodies were used, and the signals were obtained and quantified using an Odyssey CLx imager (LI-COR Biotechnology) and Image Studio Lite software (version 5.2). Actin was used as an internal loading control. All Western blots for PDGFRA, with the exception of the experiment shown in A, were performed in a blinded manner in which the investigator was not aware of the identities of the samples. [...] For confocal laser-scanning microscopy, HEK293 cells were grown on No. 1.5 glass coverslips coated with 25 μg/ml poly-d-lysine hydrobromide (Sigma-Aldrich Sweden AB), followed by transfection with the indicated plasmids. Forty-eight hours after transfection, cells were fixed with 4% paraformaldehyde as described previously () followed by permeabilization and blocking with PBS containing 5% FBS and 0.05% Tween 20. Cells were incubated with primary and secondary antibodies in PBS containing 5% FBS and 0.05% Tween 20 for 1 h at ambient temperature with washes between the incubations. Prolong Gold (Thermo Fisher Scientific, P36930) with DAPI was used to mount the samples and counterstain nuclei; 3D images were acquired at ambient temperature with a Zeiss LSM 710 confocal microscope equipped with a plan-apochromat 63 × 1.4 numerical aperture objective controlled by ZEN 201 SP1 software (Carl Zeiss AB, Stockholm, Sweden). Pixel size was set according to the Nyquist criterion. For quantification of PON2 expression, the fields of view were exclusively selected based on the DAPI nuclear staining. The mean total intensity was calculated using IMARIS software (Bitplane AG, Zürich, Switzerland). At least 500 cells were quantified. z-Stacks were displayed as maximum z-projections, and brightness and contrast were adjusted (identically for all images) using ZEN 210 SP1 software. For the co-localization analysis, z-stacks with 0.6-μm step sizes were acquired. To quantify the degree of LRIG1 and PON2 co-localization, 3D images were preprocessed, and cellular regions of interest were segmented using ImageJ (). The cellular regions of interest for each channel were used to quantify the Pearson (M1 and M2) coefficients using ImageJ with the JACoP plugin (). […]

Pipeline specifications

Software tools Imaris, ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Neoplasms
Chemicals Tyrosine