Odyssey fc 2800

Protein isolation and western blot analysis were performed as previously described [26 (link),37 (link)]. Briefly, protein was isolated by sonication of treated cancer cells or tumors in lysis buffer, followed by centrifugation. Protein concentration in isolated supernatant was quantified by a bicinchoninic acid assay (BCA) protein measurement method. Protein signals were detected by Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific) and were developed using Odyssey Fc 2800 (LI-COR Biosciences, Lincoln, NE, USA). Software ImageJ (NIH) was used quantify protein bands in Western blots. Cofilin was used as an internal protein loading control for normalizing protein signals, as cofilin’s levels were more stable than those of β-actin, under the eATP treatment.

Protein homogenates (10 μg, 20 μg for RGS10 measurements) in Laemmli buffer (Bio-Rad) were resolved on 4–20% Mini-PROTEAN TGX Precast gels (Bio-Rad) and transferred to membranes using the Trans-Blot Turbo System (Bio-Rad) according to manufacturer’s protocol. Total protein was visualized using the REVERT Total Protein Stain (LI-COR) according to manufacturer’s protocol and imaged on an Odyssey Fc 2800 (LI-COR). Blots were then cut to facilitate single exposure to multiple antibodies, washed, blocked for ~ 1 h, and incubated overnight with primary antibodies in 5% powdered milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Blots were washed and incubated one hour with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Additional file 5). Chemiluminescent signal was imaged on Azure Biosystems’ C400 system. Bands were quantified using Image Studio Lite v5.2. Gut protein levels were normalized to levels of β-actin in each sample from the same blot. β-actin was deemed an inappropriate loading control for striatum samples because its levels were not consistent across experimental groups, so levels of targets measured in striata were normalized to total protein. Full, unedited images of all blots are provided in Additional file 6.

Proteins were isolated from cells treated with no treatment,.5 mM ATP for hours or 6 hours, or 10 ng/ml TGF-β for 2 hours or 6 hours. Proteins were analyzed with western blots using appropriate primary rabbit anti-human antibodies all purchased from Cell signaling Technologies: E-cadherin (# 3195), Snail (#3879), Vimentin (# 5741), MMP-1 (#54376), MMP-3 (# 14351), MMP-9 (# 2270), Claudin-2 (# 48120), and NF-κBp65 (# 4764). Secondary antibody staining was completed with anti-rabbit IgG, HRP-linked antibody (Goat, 1:1000, CST, #7074). Cofilin (D3F9) XP^® Rabbit mAb (#5175) was used as a protein loading control. The signals were detected with Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific) and was developed using (Odyssey Fc 2800, LI-COR Biosciences). Intensities of protein bands were quantified by the corresponding Odyssey Fc software used to develop blots.