Western Blot Analysis of Secreted Proteins

Whole protein cell lysates were extracted using RIPA buffer containing protease and phosphatase inhibitors. Conditioned medium (~4 ml) was collected after each experiment, filtered through 0.2 μm pores, concentrated 40X using 10 K protein concentrators (Pierce) and stored at −80 °C until use. Protein concentration was determined using the BCA protein assay kit (Pierce) and about 20–40 μg of protein were separated on a 12% SDS-PAGE. Proteins were transferred to a PVDF membrane which was then blocked in 5% non-fat milk or BSA in TBS-T buffer and incubated with anti-Growth Differentiation Factor 15 (GDF15) (Cell Signalling), anti-phospho-Akt (Ser 473) (Cell signalling), anti-Akt total (Cell Signalling), anti-phospho-CREB1 (Ser 133) (Abcam) or anti-CREB1 total (Abcam) antibodies overnight. A Coomassie staining (Sigma), for blots with conditioned medium, or antibodies against β-tubulin or β-actin, for blots with cell extracts, were used as loading controls. The detection of antibodies was performed with enhanced chemiluminescent system (Pierce) using Kodak Biomax light films. Films were then scanned using an HP Scanjet G4010 scanner and images were analyzed in Adobe Photoshop. Color was discarded and images were converted to grayscale. In some cases contrast was used in the entire blot to enhance image clarity. No other image manipulation was performed. Uncropped images of Western Blotting used in this article can be found in Supplementary Fig. 6 as indicated in each figure legend.