Fusion sl advance

For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS) and western blotting (Laemmli 1970 (link)), GP and mock-treated cells were lysed in 1% SDS (Roth, Karlsruhe, Germany) with 0.1% diluted protease inhibitor cocktail and sonicated. Protein concentration was determined using the DC™ Protein Assay Kit. For each sample, 20 μg protein was mixed with 4xSDS-PAGE sample buffer (40% glycerol, 20% β-mercaptoethanol, 12% SDS, 0.4% bromophenol blue) and heated at 95 °C for 10 min. Subsequently, the samples were subjected to 12% or 15% (w/v), SDS-polyacrylamide gels, respectively. After blotting the proteins onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Solingen, Germany) and blocking (5% (w/v) milk powder), incubation with the primary (1:1000) and secondary antibody (1:15,000) was performed, the blot developed using the ECL-system (Cell Signaling Technology), and monitored by the Fusion SL Advance gel documentation device (Peqlab, Erlangen, Germany). Quantification of proteins was done using the FusionCapt Advance software.

For SDS (sodium dodecyl sulfate polyacrylamide gel) electrophoresis and Western Blotting (Laemmli 1970 (link)), cells were lysed after incubation with GP in 1% SDS (Roth, Karlsruhe, Germany) with 1:1000 protease inhibitor cocktail and sonicated. Protein concentration was determined with the DC™ Protein Assay Kit. 20 µg protein of each sample was mixed with 4 × SDS-PAGE sample buffer (40% glycerol, 20% β-mercaptoethanol, 12% SDS, 0.4% bromophenol blue) and heating at 95 °C for 10 min. Subsequently, the samples were subjected to 12% or 15% (w/v), respectively, SDS-polyacrylamide gels. After blotting of proteins onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Solingen, Germany), the blot was developed using the ECL-system (Cell Signaling Technology, Frankfurt a. Main, Germany) and monitored by the Fusion SL Advance gel documentation device (Peqlab, Erlangen, Germany). Quantification of proteins was done by FusionCapt Advance software.