Cnmcs compartmental protein extraction kit

The tissue and tumor samples were homogenized with a Bullet Blender (Next Advance, Averill Park, NY) according to the manufacturer’s instructions. Sequential extractions of frozen samples of tumors were performed using the CNMCS compartmental protein extraction kit (Millipore, Billerica, MA) as previously described [13 (link)]. In brief, frozen samples were homogenized and extracted sequentially to remove preferentially (1) cytosolic proteins, (2) nuclear proteins, (3) membrane proteins, (4) cytoskeletal proteins leaving a final insoluble fraction enriched for ECM proteins, although different tissues behave somewhat differently in the ease of extraction so that proteins sometimes appear in more than one fraction. The effectiveness of extraction of specific proteins was monitored by immunoblotting using the following antibodies: rabbit anti-collagen I, mouse anti-GAPDH and rabbit anti-histones (Millipore, Billerica, MA), the rabbit anti-actin antibody (serum 14–1) was generated in our laboratory.

Mouse tissues were collected and lysed with the CNMCS Compartmental Protein Extraction Kit (Millipore, 2145) according to the manufacturer’s protocol. The final pellets were then sent to the Proteomics Core at University of Texas Southwestern Medical Center. Data were acquired by the facility core and analyzed using Proteome Discoverer 2.4 (Thermo Fisher Scientific), followed by searching of the mouse protein database from UniProt. For data analysis, to increase the level of confidence for the identified proteins, the results were filtered with a threshold of 10 peptide-spectrum matches (PSMs). All the protein with more than 10 PSMs was added up and calculated as total protein abundance for WT and pNF. For each individual target, the abundance ratio was calculated by its abundance normalized to total protein abundance (abundance ratio of protein X = protein X abundance / total protein abundance). This abundance ratio for each protein was used to compare between WT and pNF. Student’s t test was used to calculate statistical difference. P less than 0.05 was considered to be statistically significant.

Normal, hyperplastic and angiogenic islets and insulinomas were homogenized with a Bullet Blender (Next Advance, Averill Park, NY) according to the manufacturer’s instructions. Decellularization (removal of intracellular proteins) and concomitant enrichment of ECM proteins were performed using the CNMCS compartmental protein extraction kit (Millipore, Billerica, MA) as previously described8 (link)32 . The effectiveness of the ECM enrichment was monitored by immunoblotting using the following primary antibodies: rabbit anti-collagen I, mouse anti-GAPDH and rabbit anti-histones (Millipore, Billerica, MA), the rabbit anti-actin antibody (serum 14–1) was generated in our laboratory.