Rabbit anti phospho mapkapk 2

Bacterial infection of the HIBCPP cells was followed by a wash step with PBS and the subsequent extraction of whole protein lysate using modified RIPA buffer (1 × RIPA lysis buffer, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, protease inhibitor cocktail). Whole protein content was determined using the DC Protein Assay (BioRad, München, Germany) according to manufacturer´s instructions. 20 µg of protein was resolved by 4–12% Bis–Tris gels (Invitrogen, Karlsruhe, Germany) and subsequently electrotransferred onto nitrocellulose membranes. Target proteins were detected using the antibodies: rabbit anti-phospho-Erk1/2, rabbit anti-rk1/2, rabbit anti-phospho-p38, rabbit anti-p38, rabbit anti-phospho-MAPKAPK-2 and rabbit anti-MAPKAPK-2 which were obtained from Cell Signalling (Cambridge, UK). To visualize immunoreactivity, the Immobilon Western Kit (Millipore, Schwalbach, Germany) was used. All blots were performed at least three times, each representing an independent experiment.

The cells were solubilized in M-PER reagent (Thermo Scientific) supplemented with protease inhibitors. The lysates were clarified by centrifugation at 16 000 × g for 30 min at 4 °C. Protein samples were loaded and separated on 6, 10 or 12% SDS-PAGE gel and immunoblotted with antibodies including mouse anti-GAPDH (sc-47724; Santa Cruz Biotechnology, 1:1000 dilution), mouse anti-Tim23 (611222; BD Biosciences, San Jose, CA, USA, 1:1000 dilution) and rabbit anti-hsp60 (12165; Cell Signalling, 1:1,000 dilution), rabbit anti-COX IV (4850; Cell Signalling, 1:2500 dilution), rabbit anti-AMPKα (5832; Cell Signalling, 1:1000 dilution), rabbit anti-phospho-AMPKα (2535; Cell Signalling, 1:1000 dilution), rabbit anti-p38 (8690; Cell Signalling, 1:1000 dilution), rabbit anti-phospho-p38 (4511; Cell Signalling, 1:1000 dilution), rabbit anti-phospho-ERK1/2 (4370; Cell Signalling, 1:1000 dilution), rabbit anti-phospho-ATF-2 (5112; Cell Signalling, 1:1000 dilution), rabbit anti-phospho-hsp27 (9709; Cell Signalling, 1:1000 dilution) and rabbit anti-phospho-MAPKAPK-2 (3007; Cell Signalling, 1:1000 dilution).