Western Blot Analysis of Protein Expression

Samples were prepared similarly as previously described^{65 (link)} with slight modifications. In short, cells were washed once in PBS, lysed in RIPA, treated with benzonase (Sigma) and rotated at 4 °C after which they were stored at −20 °C. Protein concentration estimation was done using a BCA estimation kit (Pierce). Equal concentrations of protein were prepared in 4x Lammeli buffer and boiled for 10 min before loading on an SDS-PAGE gel. Resolved proteins were then transferred onto a nitrocellulose membrane (Amersham), blocked in 5% BSA/PBS and then incubated overnight with one of the following antibodies, washed in TBS-T and followed by the respective secondary Ab for 2 h. γ-tubulin was used last as a loading control: anti-Pdk4 (1:1000, Abcam, ab38242), anti-Serpinf1 (1:2000, Sigma-Aldrich, AV20020-50UG), anti-γ-tubulin (1:1000, Sigma-Aldrich, T6557), ECL anti-rabbit IgG-HRP (1:5000, GE Healthcare), ECL anti-sheep IgG-HRP (1:5000, GE Healthcare). Blots were developed with ECL substrate (Pierce) and the signal detected on a Fusion-FX7 Chemiluminescence detection system (PeQlab). Originals are provided in Supplementary Fig. S4. Each experiment involved biological triplicates, which were subsequently used for statistical testing.