Cocktail set 2

SILAC-labelled BMDCs were first incubated in D-MEM (1.9 mM Ca²⁺) for 2 hours at 37 °C. ‘Light’ isotope-labelled cells (¹²C6-arginine/¹²C6-lysine) were next treated for 10 or 30 minutes at 37 °C with 100 ng/ml of either CyaA toxin or CyaA-AC^- toxoid dissolved in TUC buffer (50 mM Tris-HCl, 8 M urea, 2 mM CaCl₂, pH 8). Corresponding ‘heavy’ isotope-labelled cells (¹³C6-arginine/¹³C6-lysine) were treated by TUC buffer alone and served as controls for both CyaA and CyaA-AC^--treated BMDCs (Supplementary Fig. S1). The whole experiment was performed in biological triplicate and SILAC groups were swapped in one replicate. Cells were next washed in ice-cold PBS and lysed in Lysis buffer (50 mM NH₄HCO₃, 1% (w/v) sodium deoxycholate (SDC)) containing a cocktail of phosphatase inhibitors (cocktail set II; Merck). Lysis was accomplished by placing the cell suspension into a boiling water bath for 5 min^{13 (link)}. After cooling of the lysed suspension to room temperature, the samples were subjected to benzonase treatment (Sigma) for 1 h and cell debris was removed by centrifugation (14,000 g, 10 min and 4 °C). Protein concentrations in supernatants were measured using the Micro BCA kit (Thermo Pierce) and the corresponding ‘light’ and ‘heavy’ isotope-labelled lysates were mixed at a 1:1 ratio based on their protein content.

Cells were lysed at indicated times p.i. in RIPA buffer supplemented with EDTA-free complete protease inhibitor mixture (Roche, Basel, Switzerland) and phosphatase inhibitors (Cocktail set II, Merck, Darmstadt, Germany). Total protein concentration was determined using a bicinchoninic acid assay. Protein samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA). Blots were blocked with 5% milk and incubated with different primary antibodies overnight, followed by incubation with a secondary antibody conjugated with horse-radish peroxidase (Agilent Dako, Santa Clara, CA, USA). Proteins were detected using a BM Chemiluminescence Blotting Substrate (POD) (Roche) or SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) on X-ray films.
Primary antibodies anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (Erk 1/2), anti-p38MAPK, anti-phospho-p-38 MAPK, anti-JNK2, anti-phospho-SAPK/JNK, anti-IκBα, and anti-phospho-IκBα were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-IL-1β and anti-phospho-IL-1β were purchased from Abcam (Cambridge, UK); anti-Actin and anti-GAPDH for loading controls were purchased from Sigma-Aldrich.