Protein biochemistry

2.2.2.1 Preparation of complete host cell protein extracts

For determining the impact of T. gondii infection on the expression and activation of STAT1, complete host cell protein extracts were prepared. To this end, two different methods were applied. To solubilise and isolate proteins, a lysis buffer with mild detergent was used. Briefly, after experimental treatment of cells as specified in the results section, cells were harvested and washed with ice-cold wash buffer (1 mM Na3VO4 in PBS). After centrifugation for 5 minutes at 400 x g at 4°C and resuspension in a defined volume of 1 mM Na3VO4 in PBS, the number of viable cells was determined using a haemocytometer grid. After centrifugation, cells were then resuspended at 7.5 x 10⁴ cells/µl of ice-cold lysis buffer (1%

(v/v) Triton X-100, 0.15 M NaCl, 50 mM Tris pH 8, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM Na3Vo4, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM EDTA pH 8) and incubated for 1 hour on ice. During incubation, samples were vigorously vortexed every 15 minutes.

After centrifugation for 5 minutes at 12,000 x g at 4°C, the supernatants were stored as complete cell protein extracts at - 80°C until further use.

Alternatively, complete cell protein extracts were prepared by cell sonoporation in harsh detergent-containing buffer. Briefly, after experimental treatment of cells as specified in the results section, cells were harvested and washed with ice-cold wash buffer (1 mM Na3VO4 in PBS). After centrifugation for 5 minutes at 400 x g at 4°C and resuspension in a defined volume of PBS, 1 mM Na3VO4, the number of viable cells was determined using a haemocytometer grid. Cells were centrifuged for 30 seconds at 12,000 x g at 4°C and then resuspended at 4 x 10⁴ in SDS-sample buffer (62.5 mM Tris pH 6.8, 6% (v/v) glycerol, 0.5% (w/v) DTT, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) supplemented with 1 mM Na3Vo4. To effectively disrupt the cells, samples were placed in ice-water slurry supplemented with common table salt and subjected to sonication (Branson Sonifier 250). The impulse was applied for 15 seconds with a duty cycle of 30% and an output level of 1. Thereafter, the samples were stored at - 80°C. Prior to protein separation by SDS-PAGE (see section 2.2.2.3), samples were denatured for 5 minutes at 99°C and centrifuged for 5 minutes at 14,000 x g at 4°C.

2.2.2.2 Preparation of cytosolic and nuclear protein fractions

To analyse the impact of T. gondii infection on the subcellular distribution of STAT1 and the DNA binding activity of nuclear STAT1, cytosolic and nuclear protein fractions were prepared.

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In most of the experiments, cells were fractionated as described previously (Lang et al., 2012) with slight modifications. Briefly, after experimental treatment of cells as specified in the results section, cells were harvested and washed with ice-cold wash buffer (0.1 mM Na3VO4 in PBS). After centrifugation for 5 minutes at 400 x g at 4°C and resuspension in a defined volume of 1 mM Na3VO4 in PBS, the number of viable cells was determined using a haemocytometer grid. For Western blot analysis cells were resupended at 4 x 10⁴ cells/µl in ice-cold lysis buffer (10 mM HEPES pH 7.8, 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 0.1 mM Na3VO4) and for EMSA experiments at 7 x 10⁴ cells/µl.

The samples were then incubated 15 minutes on ice before adding 0.6% (v/v) Nonidet-P40. Thereafter, samples were vigorously mixed and complete cell lysis was confirmed microscopically by staining a sample aliquot with an equal volume of 0.1% (w/v) trypan blue. After centrifugation for 30 seconds at 12.000 x g at 4°C, the supernatant was stored as cytosolic protein fraction at - 80°C or in case of EMSA experiments was discarded. The pellet containing nuclei and other larger organelles was washed with lysis buffer. Finally, for Western blotting analysis cells were resuspended at 2 x 10⁵ cells/µl in ice-cold nuclear protein extraction buffer (50 mM HEPES, 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 0.1 mM Na3VO4) and for EMSA experiments at 5 x 10⁵ cells/µl. After incubation for 20 minutes at 4°C under constant rotation, the samples were centrifuged for 5 minutes at 12.000 x g at 4°C. The supernatant was stored as nuclear protein fraction at - 80°C.

For experiments with protein extracts from STAT1-reconstituted U3A cells, subcellular fractionation was modified and performed as described previously (Riebeling et al., 2014). Briefly, after experimental treatment of cells as specified in the results section, cells from a well of a 6 well plate were rinsed twice with PBS and directly lysed for 5 minutes in the cell culture vessel using 60 µl ice-cold cytosolic protein extraction buffer (20 mM HEPES, 10 mM KCl, 1 mM EDTA, 0.1 mM Na3VO4, 10% glycerol, 0.1% IGEPAL CA-630, 1X protease inhibitor cocktail (Roche), 3 mM DTT and 0.4 mM Pefabloc (Roche), pH 7.4). Cells were then scraped off and centrifuged for 15 seconds at 16,100 x g at 4°C to pellet heavy organelles including nuclei. After transferring the supernatant into a new tube, it was further cleared by centrifugation for 5 minutes at 16,100 x g at 4°C and collected as cytosolic protein fraction. The pelleted nuclei were then incubated in 60 µl of ice-cold nucleic protein extraction buffer (20 mM HEPES, 420 mM KCl, 1 mM EDTA, 0.1 mM Na3VO4, 20% glycerol, 1X protease inhibitor cocktail, 3 mM DTT, 0.4 mM Pefabloc, pH 7.4) for 30 minutes on ice. The extract was then centrifuged for 15 minutes at 16,100 x g at 4°C and the supernatant collected as nuclear protein fraction. Finally, cytosolic and nuclear fractions were pooled as complete cell protein extract and stored at - 80°C until further use.

2.2.2.3 SDS-polyacrylamide gel electrophoresis

Proteins in cell lysates and in oligonucleotide-precipitated samples were separated according to their size by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

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To this end, two types of gels were combined, which differed in their pore size. First, the separating gel solution (0.375 M Tris pH 8.8, 0.2% (w/v) SDS, 10% (v/v) acrylamide/ 0.27% (v/v) bisacrylamide, 0.04% (w/v) APS and 0.2% (v/v) TEMED) was poured into a vertical gel cassette. The gel was carefully overlaid with isopropanol and allowed to completely polymerise. After removing the isopropanol and rinsing the gel surface with water, the stacking gel solution (0.125 M Tris pH 6.8, 0.2% SDS, 5% (v/v) acrylamide/ 0.13% (v/v) bisacrylamide, 0.04% (w/v) APS, 0.4% (v/v) TEMED and 0.01% (w/v) bromophenol blue) was poured onto the separating gel and a comb was inserted.

Protein extracts were mixed with SDS-sample buffer to a final concentration of 62.5 mM Tris pH 6.8, 6%

(v/v) glycerol, 0.5% (w/v) DTT, 2% (w/v) SDS and 0.01% (w/v) bromophenol blue and were denaturated for 5 minutes at 95°C. Pre-stained marker proteins were likewise treated in parallel. Proteins were then separated at 25 mA per gel (8 cm wide, 0.75 mm thick) in SDS-running buffer (25 mM Tris, 192 mM polyacrylamide gel onto a nitrocellulose (NC) membrane. The electro-transfer was carried out using a semi-dry blotting device. To this end, a transfer sandwich was assembled in the following order (from anode to cathode): 6 Whatman filter papers (soaked in 0.3 M Tris pH 10.4 and 20% (v/v) methanol), 3 Whatman filter papers (soaked in 25 mM Tris pH 10.4 and 20% (v/v) methanol), NC membrane (soaked in 25 mM Tris pH 10.4 and 20% (v/v) methanol), SDS-polyacrylamid gel and 9 Whatman filter papers (soaked in 40 mM 6-aminocapronic acid pH 7.6 and 20% (v/v) methanol). The transfer was performed for 90 minutes at 0.8 mA per cm² of gel. Afterwards, proteins were stained unspecifically using Ponceau S to control the transfer efficacy. Unspecific binding sites on the NC membrane were then blocked for 1.5 hours with 5% (w/v) skimmed milk in TBST (0.9% (w/v) NaCl, 10 mM Tris, 0.1% (v/v) Tween-20, pH 7.4). Thereafter, the blot was washed with TBST before being incubated with the primary antibody (Table 25) overnight at 4°C. After having been washed repeatedly with TBST for 5, 10 and 20 minutes, the membrane was then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Table 26) for 1.5 hours. Thereafter, the membrane was washed two times in TBST for 10 minutes each and once for 20 minutes. All washing and incubation steps were carried out under gentle shaking. Enhanced chemiluminescence (ECL) detection (Amersham GE Healthcare) was used to detect bound antibodies. To this end, the ECL detection reagent was prepared as recommended by the manufacturer and poured onto the membrane. After 1 minute, superfluous solution was removed and the membrane was covered with transparent foil. Chemiluminescence was detected using the LAS-4000 (FUJIFILM) gel documentation device and the Image Reader LAS-4000

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software. To subsequently detect another specific protein on the same membrane, bound antibodies were stripped off from the blot. To this end, the membrane was washed in ddH2O for 5 minutes, then incubated in 0.2 mM NaOH for 5 minutes and washed again twice with ddH2O. After re-blocking, the membrane was re-probed with another primary antibody.

Table 25: Primary antibody solutions for Western blot analysis

Antibody Dilution concentration Incubation buffer

Mouse anti-pan Actin (Clone C4) 1:5000 5% BSA (w/v) in TBST, pH 7.4 Rabbit anti-Brg-1 (H-88) 2µg/ml 5% BSA (w/v) in TBST, pH 7.4 Rabbit anti-GAPDH (14C10) 1:1000 5% BSA (w/v) in TBST, pH 7.4 Rabbit anti-phospho-Stat1(pS727) 1:1000 5% BSA (w/v) in TBST, pH 7.4 Mouse anti-phospho-Stat1 (pY701)

(Clone 14/P-STAT1 (RUO))

0.5 µg/ml 1% BSA (w/v), 10 mM Tris, 0.1 M NaCl, 0.1% (v/v) Tween-20, pH 7.4 Rabbit anti-STAT1α p91 (M-23) 1 µg/ml 5% BSA (w/v) in TBST, pH 7.4

Table 26: Secondary antibody solutions for Western blot analysis

Antibody Dilution concentration Incubation buffer

Donkey IgG anti-Rabbit IgG (H+L)-HRPO 1:2000 The incubation solution always corresponded to the primary antibody's one.

Goat IgG anti-Mouse IgG (H+L)-HRPO 0.2 µg/ml

2.2.3 Nucleic acid analysis