Tina 2

COS-7 cells transfected with pDSRed2-humanTau (2N4R isoform) and cultured in DMEM/5% fetal calf serum for 24 h in 12-well culture plates were incubated with the respective compounds each 8 µM or 0.1% DMSO as vehicle control for 12 h at 37 °C. Cells were scraped, centrifuged for 10 min at 4 °C with 300× g, washed with cooled PBS and centrifuged again. Proteins were extracted with 300 µL PBS, 1% Triton X-100 including protease and phosphatase inhibitors (cOmplete^TM, PhosSTOP^TM, Roche Life Science, (Germany, Penzberg). Protein content was calculated by the Pierce 660 nm protein assay (Thermo Scientific, Rockford, IL, USA). Ten micrograms of protein from each sample were run as duplicates on 8% SDS-PAGE and transferred to a PVDF membrane. Total tau protein was labelled with a polyclonal total tau antibody (1:4000, v:v, #A0024, Dako, Glostrup, Denmark) and phospho-tau (pThr231, pSer235) was labelled with the monoclonal antibody AT180 (1:500, v:v, MN1040, Thermo Scientific). Western blots were imaged after incubation with anti-rabbit HRP/anti-mouse HRP (1:10,000, v:v NA934, NA931, GE Healthcare, Little Chalfont, UK) using the DNR Chembis ECL station and immunoreactive bands were quantified by densitometry using Tina 2.09 software (Raytest, Straubenhardt, Germany). Phospho-tau immunoreactivity was normalized to total tau immunoreactivity.

Primary alveolar epithelial cells were isolated and cultivated as described above. SDS-PAGE and Western blot were performed as described earlier [35 ]. The following antibodies were used: Claudin-18 (38–8000, ThermoFisher, 1:500), SP-C (ab90716, Abcam, 1:500), AQ5 (ab92320, Abcam, 1:500), and a-Tubulin (ab89984, Abcam, 1:1000), anti-rabbit-HRP (P0448, DakoCytomation, Glostrup, Denmark, 1:1000), and anti-chicken-HRP (ab97135, Abcam, 1:1000). HRP-activity was visualized with Clarity Western ECL substrate (BioRad, Hercules, USA). The membranes were developed on conventional films in a dark room and analyzed densitometrically after scanning with TINA 2.0 (Raytest GmbH, Germany).

To probe for STAT1 DNA-binding activity in T. cruzi-infected cells, electrophoretic mobility shift assays (EMSAs) were performed. Five microliters of cellular extracts were incubated with 1 ng of [³³P]-labeled M67 duplex oligonucleotide [39] (link). The radioactively labeled probe, which was generated by an end-filling reaction using the Klenow fragment (New England Biolabs), contained a consensus STAT1-binding site (5′-CGACATTTCCCGTAAATCTG-′3; GAS sequence underlined, 4 bp overhangs at the 5' end and the respective antisense oligo are not shown). For competition experiments, cellular extracts were first incubated with [³³P]-labeled M67 in EMSA buffer for 15 min at RT, and then a 750-fold molar excess of unlabeled M67 DNA was added. In supershift assays, 20 ng of either the STAT1-specific antibody C-24 or a non-specific STAT3 antibody were present in the shift reaction. The reactions were loaded on a 4% 29∶1 acrylamide∶bisacrylamide gel at 4°C and separated at 400 V. DNA-binding activity of STAT1 was visualized on vacuum-dried gels with a phosphoimaging system (FLA-5100, Fuji) using the programs Aida Image Analyzer v.4.06 and TINA 2.0 (Raytest).