Mammalian protein extraction reagent lysis buffer

PamGene serine-threonine kinase (STK) multiplex activity assays were used to investigate STK activity. Murine cardiac fibroblasts were treated with vehicle or Yoda1 for 10 min, collected, and lysed using mammalian protein extraction reagent lysis buffer (Thermo Fisher Scientific) containing Halt phosphatase and Halt protease inhibitor cocktails (Pierce) for 30 min on ice. A protein quantification assay was then performed as stated above, and samples were snap-frozen in liquid nitrogen. 1.2 μg of protein was loaded onto each STK PamChip array. Phosphorylation of PamChip peptides was monitored by PamStation 12 following the manufacturer's protocols as described previously (40 (link)). Signal intensities were analyzed using PamGene's BioNavigator software as Yoda1-treated versus DMSO treatment after 10 min. Permutation analysis resulted in a specificity score (mapping of peptides to kinases) and a significance score (difference between treatment groups) for each kinase. The combined score was used to rank and predict top kinase hits.

Cells were lysed with a Mammalian Protein Extraction Reagent lysis buffer (ThermoFisher Scientific) containing a protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Santa Cruz). Proteins were separated according to the protein size by a 10% or 15% SDS‐PAGE and transferred onto a nitrocellulose membrane (ThermoFisher Scientific). Membranes were blocked for 1 h at room temperature in TBST containing 10% milk powder (Roth) followed by overnight incubation with primary antibodies diluted in TBST containing 5% bovine serum albumin (Roth) at 4 °C. For detection, horseradish peroxidase‐conjugated secondary antibody (Cell Signaling) diluted in TBST containing 5% milk powder was used in combination with enhanced chemiluminescence (ECL) solution to detect the bands via a Chemidoc XRS⁺ (BioRad). For statistical analysis, the intensity of the bands was measured via ImageJ and the quantification of STAT3 activation corresponded to the ratio of P‐STAT3 on total STAT3 levels after normalization to their GAPDH bands and the quantification of SOCS3 corresponded to the ratio of SOCS3 levels after normalization to GAPDH levels.