Sodium orthovanadate

Cells were lysed by lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 1 mM sodium orthovanadate (New England Biolabs, Beverly, MA) plus a mixture of protease inhibitor cocktail (Sigma) for 10 min on ice, then sonicated and the cell debris was removed by centrifugation (18,000×g for 30 min). Bicinchoninic acid (BCA, Pierce, Rockford, IL) assay was preformed to determined protein concentrations. Cells lysates was precleared by incubation with Protein A/G beads (Sigma) for 1 h at 4 °C to eliminate nonspecific binding. The lysates were then subjected to inmmunoprecipitation overnight at 4 °C with indicated antibodies or purified mouse/rabbit immunoglobulin G (Sigma), followed by incubation with Protein A/G beads for 2 h. Precipitates were washed 4 times with a lysis buffer before resuspending with a 2× protein sample buffer, and were boiled for 5 min to release the bound proteins. Subsequently, the proteins were analyzed by Western blot analysis with the indicated antibodies.

Cells were lysed into lysis buffer containing 1% Na deoxycholate (Sigma), 150mM NaCl, 10mM PO₄Na₂/K, pH 7.2 and supplemented with inhibitor cocktail (Sigma), 2mM EDTA, 1mM sodium orthovanadate (New England Biolabs), phosphatase inhibitor (Thermo Fisher) and 5mM PMSF (phenylmethanesulfonyl fluoride, Sigma). Benzonase nuclease (Santa Cruz) was added to reduce viscosity in protein extracts. Protein content was measured using BCA method (Biorad). Each sample was dissolved in Laemmli 5X buffer, and then denatured at 100°C for 10min. After SDS-PAGE and transfer onto nitrocellulose membrane (Hybond, GE Healthcare), membranes were incubated with goat polyclonal primary antibodies against p-AMPk (1/1000, Millipore) or AMPk (1/1000, Sigma) overnight at 4°C. Membranes were incubated with anti-goat peroxidase-conjugated anti-IgG secondary antibody (1/5000, Santa Cruz), developed using ECL substrate solution, exposed to the Fusion Fx7 (Thermo Fisher) and analyzed using Quantity One software (Biorad).

Mitoxantrone, rhodamine-123, Ko143, ammonium molybdate, potassium antimony (III) tartrate hydrate, adenosine 5′-triphosphate disodium salt (ATP), ascorbic acid and ouabain octahydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). PEI (Poly-ethyleneimine) is obtained from Polysciences Europe (Eppelheim, Germany). G418 is from Santa Cruz Biotechnology (Dallax, TX, USA). Sodium orthovanadate is from New England Biolabs (Frankfurt, Germany). Oligonucleotides for site-directed mutagenesis were from Eurofins (Munich, Germany). Monoclonal mouse anti-ABCG2 (BXP-21) was purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-β-actin (D6A8) was from Cell Signaling (Danvers, MA, USA). All other chemicals were of molecular biology grade from Sigma-Aldrich (St. Louis, MO, USA). Phusion DNA polymerase and DpnI are from NEB (New England Biolabs, MA, USA). Plasmids templates using in this study, pcDNA3.1(-)-hABCG2 (wild type) and pEGFPC1-hABCG2 (wild type) were kindly provided by Balázs Sarkadi (Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary).

Skin biopsies were snap frozen in Precellys Soft tissue homogenizing CK14 tubes (Bertin instruments, Montigny-le-Bretonneux, France) and lysed with a Precellys 24 tissue homogenizer (Bertin instruments, Montigny-le-Bretonneux, France) in Cell signaling lysis buffer (Cell Signaling Technology, Leiden, The Netherlands) added Complete Mini Protease Inhibitor Cocktail Tablet (Roche diagnostics, Mannheim, Germany), Halt^TM phosphate inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and Sodium Orthovanadate (New England Biolabs, Ipswich, MA, USA). The protein concentration was measured by the use of BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) and adjusted to 2 μg/μl. Human TNF-α, IL-17A, IL-17AF, IL-22, IL-23, IL-1β and IL-6 and murine TNF-α, IL-17A, IL-17AF, IL-22, IL-23, IL-1β, IL-6 CXCL-1 and MIP-3α were analyzed in skin lysates by the MSD platform (Meso Scale Diagnostics, Rockville, MD, USA) according to the manufacturer´s instructions. In addition, the protein levels of murine TNF-α, IL-1β, IL-17A, IL-17AF, IL-22, IL-23 and IL-6 were analyzed in serum by the MSD platform. Standard curves were prepared with a matrix relevant for the sample and no cross-reactivity to mouse and human proteins were shown in the assays except from the murine IL-22 assay.

A total of 5.3 × 10⁵ cells were seeded in 6-well plates and cultured for 24 h. Cells were infected with C. trachomatis (0.7 IFU/cell) and centrifuged at 700 × g for 1 h at 35°C. Infected cells were incubated at 37°C under a 5% CO₂ atmosphere for 24 h. Afterwards, they were treated with or without 25 μM sodium orthovanadate (New England BioLabs GmbH) for 3 h.