Western Blot Protein Detection Protocol

The conditioned media derived from 12-well plates were concentrated approximately five times using Amicon centrifugal filter device (Millipore, Danvers, MA, USA; 3 kDa MWCO), and then used for Western blot analysis as previously described [27 (link)]. An equal amount of each sample was resolved on an SDS-polyacrylamide gel and electroblotted onto a PVDF membrane. The blots were blocked with 5% skim milk in PBS containing 0.05% Tween 20 and incubated with an appropriate antibody (diluted 1:1000 in blocking solution). Immobilon Western HRP Substrate kit (Merck, Kenilworth, NJ, USA) and ImageQuant LAS 4000 software (GE Healthcare, Chicago, IL, USA) were used to detect Chemiluminescence signals. Band intensities were quantified using ImageJ software. One representative Western blot for each experiment is shown in the figures. Data from three independent experiments were averaged for each condition and presented in the form of bar graphs. Data are expressed in fold changes compared with control (mean ± SD). Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA), and p-values were calculated by paired t-test.