Dataset for "Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand"

Data collection method:

Samples were generated using methods discussed in paper manuscript "Membrane Extraction with Styrene-Maleic Acid Copolymer Results in Insulin Receptor Autophosphorylation in the Absence of Ligand." Samples were separated on 10% SDS polyacrylamide gels and transferred onto nitrocellulose using a semi-dry transfer apparatus, before immunodetection with primary antibodies; either total antibodies or phopho-specific antibodies. Blots were incubated with secondary antibody,HRP, followed by washes. ECL™ Select Western blotting detection reagent was used for detection. Images were acquired with EPI Chemi II darkroom (UVP).

Technical details and requirements:

Quantification of bands on immunoblots was measured by densitometry using Image Studio Lite (LI-COR Biosciences®). Specific band intensities were normalized to total Akt levels for that sample. The ratios between phospho-specific and total protein band intensities were calculated in the same sample. Data has been deposited and can be opened with Microsoft Excel.

Additional information:

A READ_ME.txt file is included to provide further information of the data present in the .zip file. Folders labelled "Figure_X", where X corresponds to the figure number, contain raw .tiff files used for representative blots in the paper. An additional MS PowerPoint file is included to annotate these images. "SX" describes the supplementary figures. All_blots_for_quantification folder contains all raw .tiff files obtained and included in quantification of densitometry values. Experiments_used_for_each_graph.xlsx highlights the experiments and bands used for each quantification, representated by graphs in the paper. The Excel spreadsheet "collated_InsR_values_for_archive.xlsx" has three tabs; 1. Densitometry_values - holds the raw densitometry values calculated with Image Studio Lite (LI-COR Biosciences®); normalisation to Akt in separate column (L); representative western blots for each sample measured and "fold difference" between basal and + Insulin samples. 2. Calculations - taking the fold difference tables for each experiment, with additional tables calculating fold difference relative to POST SMA Basal (Green) and PRE RIPA Basal (Orange). Averages were taken for each sample across multiple experiments and standard deviation (SD) and standard error mean (SEM) were caculated. 3. Stats - Statistic for each protein of study; InsR, IRS and Akt, across multiple experiments using paired t-tests.