Western blot analysis of sentinel proteins - inhibition studies

We selectively inhibited the studied proteins one at a time and measured how a particular inhibition affected the activities of all sentinels. Inhibitors were added to starvation medium 2 h prior to ligand-induced stimulation. They were also included in the treatment medium, i.e., cells were exposed to respective inhibitors continuously before and after the stimulation with ligands. The following inhibitors and doses were used: U0126 (Erk1/2 inhibitor; 20 μM), JNK Inhibitor VIII (JNK inhibitor; 20 μM), Stattic (STAT3 inhibitor; 10 μM), Akt Inhibitor VIII (Akt inhibitor; 10 μM), and Birb-796 (p38 MAPK inhibitor; 5 μM). No-ligand measurements where cells were treated with minimal media provided the negative control.

Lysates for Western blotting studies were collected 15 min after activation. Data for the 184A1L5 cells was collected earlier (Gong et al. 2015). To collect the data anew for the MDA-MB-231 cells, plates were washed with ice-cold PBS and cells were then immediately lysed using RIPA buffer prepared with protease and phosphatase inhibitors. Plates were lysed over a period of 30 min, on ice, prior to cell scraping and transfer of the lysate to microtubes. Lysates were centrifuged at 12,000 rpm for 10 min. The supernatant was aspirated into fresh microfuge tubes for analysis and the pellet was discarded. Lysates were stored at −80 °C between analyses. Total protein concentration of cell lysates was determined by the Bicinchoninic Acid assay technique (reagents from Sigma). Total protein amount was matched to 20 μg protein/well and loaded to SDS-PAGE gels. TGX precast 10% gels from Bio-Rad with 10 wells were used. Electrophoresis was done using the Bio-Rad Mini-Protean Tetra System and proteins were transferred from the gel to PVDF membranes using the Bio-Rad Trans-blot Turbo transfer system. PVDF membranes were developed using ECL. Western blot images were captured by a LI-COR C-DiGit scanner. Images were analyzed using the Image Studio Digits v3.1 software.

The Western blots (Fig. ​(Fig.1)1) illustrate that the chosen inhibition conditions effectively block phosphorylation of their designated targets, thus meeting the perturbation requirements necessary to infer regulatory interactions with MRA and BVSA. We note that our studies require comparison between cell lines and stimulation conditions; i.e., data from multiple plates or gels need to be combined during analysis. To increase the quality of merging data from multiple gels, we used “common positive controls” (CC; cf. Fig. ​Fig.1),1), which are measurements for a strong signal condition that are performed on every gel in one or more wells. The lysate material used for CC is prepared in large batches at once and same lysate is later used on every gel. Measured CC values are then used for quality checks and normalization of gels against each other.

Representative Western blots used in the cell line comparison study for the (a, top) HME and (b, bottom) MDA-MB-231 cells. Cells were activated for 15 min with 12 ng/ml EGF and 40 ng/ml HRG. Labels on the right indicate the measured phospho-protein. Labels below blots indicate the treatment conditions used in the study: C− (negative control), C+ (positive control), CC (common control, used for normalization between gels, analogous to the positive control condition. Loading material used for the two CC lanes were from different batches.), i-Akt (Akt inhibition), i-Erk (Erk inhibition), i-p38 (p38 inhibition), i-JNK (JNK inhibition), and i-S3 (STAT3 inhibition). Data for the PI3K inhibition (i-P3K) was not used in the analysis. Note that the layout of the gels is slightly different for the two cell types. Loading control measurements using β-actin are shown for illustration purposes