Number of cycles Temperature Time Step
1x 90 C 1 min Initial denaturation
96 C 10 s Denaturation
25x 55 C 5 s Primer annealing
60 C 4 min Elongation
1x 4 C 1 Cooling
3.4 Biochemical methods
3.4.1 Preparation of cell lysates
To prepare cell lysates, cells were washed with ice-cold PBS and collected by scraping from the cell culture dish. Cells were pelleted and resuspended in designated lysis buffer sup-plemented with protease- and phosphatase-inhibitor. Lysis was performed for 30 min on ice with recurrent resuspension. Lysates were centrifuged for 10 min at 15.000 g and cleared supernatants were transferred to a new reaction tube. Following lysis buffers were used:
GFP-Trap-Lysis buffer for GFP-trap experiments and detection of cellular and viral proteins, Kinase-binding buffer for PKR-immunoprecipitation experiments.
3.4.2 SDS PAGE
Proteins can be separated according to their molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Polyacrylamide gels were prepared in two steps employing the Mini-PROTEAN®3 gel casting equipment (Biorad). First, the separation gel was prepared. After complete polymerisation, the stacking gel was produced in a second step and lanes were generated by insertion of a plastic comb with the designated number of lanes. For a 1.5 mm gel, 10 ml of separation gel with desired concentration and 5 ml of a 5 % stacking gel were generated according to table 3.1. Samples were prepared by mixing with 6x sample buffer and incubation at 95 C for 5 min. Samples were loaded and proteins were separated in a vertical gel chamber (Biorad) in SDS-running buffer by application of an electric field at a constant current of 25 mA per gel.
3.4.3 Coomassie staining of polyacrylamide gels
Separated proteins in polyacrylamide gels can be visualized by staining with Coomassie Brilliant Blue R-250 dye, which unspecifically binds to basic amino acid side chains.
In a first step, proteins were fixed in the gel with fixing solution consisting of 3 % ethanol and 10 % acetic acid for 20 min RT. Then, proteins were stained with Coomassie Brilliant
separation gel stacking gel
7.5 % 10 % 12.5 % 5 %
30 % Acrylamide/Bisacrylamide (29:1) 2.5 ml 3.3 ml 4.1 ml 0.83 ml
ddH2O 4.8 ml 4.0 ml 3.2 ml 2.8 ml
1.5MTris/Cl pH 8.8 2.5 ml
-0.5MTris/Cl pH 6.8 - 1.25 ml
10 % SDS/ 100 l 50 l
10 % APS/ 100 l 50 l
TEMED 6 l 6 l
Table 3.1. Composition of SDS polyacrylamid gels. Composition of SDS polyacrylamid gels with different acrylamid concentrations is given. Amounts are sufficient for one gel of 1.5 mm.
Blue R-250 solution for at least 1 h at RT, followed by removal of unbound dye by repeated washing of the gel with 10 % methanol.
3.4.4 Western transfer and immunoblot analysis
To specifically detect separated proteins with corresponding antibodies, proteins were trans-ferred on a nitrocellulose membrane by semi-dry western blotting. The Trans-Blot®SD Semi-Dry Transfer Cell apparatus (Biorad) was used with the following setup: Anode, 2x Whatman paper, SDS acrylamidgel, nitrocellulose membrane, 2x Whatman paper, cath-ode. Proteins were transferred from the acrylamid gel to the nitrocellulose membrane by applying a constant current of 75 mA per gel for approximately 80 min (for a 1.5 mm gel).
Membranes were blocked with a solution of 3 % milk powder in 1x TBST or 5 % bovine serum albumin (BSA)/TBST (for detection of phospho-proteins) for at least 30 min RT. Primary an-tibodies were diluted in 0.5 % milk powder in TBST or 1 % BSA/TBST (for phospho-proteins) and membranes were incubated with antibody dilutions ON at 4 C with gentle shaking to prevent membranes from running dry. Membranes were washed with 1x TBST for at least three times before incubation with secondary horse raddish peroxidase (HRP)-coupled anti-bodies, diluted in 0.5 % milk powder in TBST or 1 % BSA/TBST (for phospho-proteins) for 1 h RT. Membranes were washed six times or more with TBST before visualisation of staining using an enhanced chemiluminiscence protocol with the SuperSignal™ West Dura Extended Duration Substrate (Thermo Scientific). Luminescence was detected using CL-XPosure™
x-ray films developed with the Curix 60 processor (Agfa) or with the Advanced Fluorescence Imager (INTAS) operated with the corresponding ChemoStar software (INTAS).
3.4.5 Coimmunoprecipitation analysis
To analyse protein-protein-interactions of endogenous PKR and binding partners, Coim-munoprecipitation experiments were conducted in non-infected and infected cells.
46 3.4. Biochemical methods
For one experiment, 7 106A549 cells were seeded in a 10 cm-dish one day prior to infection with influenza A/PR/8 WT, NS1 or R46A virus (see section 3.2.1) at an MOI of 1.5. 16 h p.i., cells were lysed with 1 ml kinase-binding buffer (see section 3.4.1) and lysates were pre-cleared with 30 l Protein G Agarose rotating for 3 h at 4 C. 100 l of cell lysates were mixed with 6x SDS sample buffer and stored at 20 C as a sample for whole cell lysate (WCL).
Meanwhile, coupling of PKR-specific primary antibody to Protein G Agarose was conducted.
For one reaction, 50 l Protein G Agarose were incubated with -PKR-antibody or -Myc-antibody (as negative control) and 500 l kinase-binding buffer rotating for 3 h at 4 C. Pre-cleared cell lysates and antibody-coupled Protein G Agarose were combined and incubated rotating ON at 4 C. To remove non-bound proteins, Protein G Agarose was washed three times with kinase-binding buffer by centrifugation for 3 min at 2000 g. Immunoprecipitated proteins and interaction partners were eluted by incubation with 2x SDS sample buffer for 10 min at 95 C. Samples of WCL and immunoprecipitate (IP) were analysed by SDS PAGE and immunoblotting (see section 3.4.2, section 3.4.4).
3.4.6 GFP-Trap
®-analysis
To analyse the interaction of green fluorescent protein (GFP)-tagged proteins with endoge-nous or co-transfected proteins, GFP-tagged proteins and binding partners were precipitated using a GFP-Trap®-matrix (Chromotek) according to the manufacturer’s instructions.
Briefly, 1 106293T cells were transfected with 1 g pEGFP-C1-PKR or pEGFP-C1-KSRP using Lipofectamine®2000 (see section 3.1.2) for 30 h at 37 C, 5 % CO2, followed by infection with influenza A/PR/8 WT, NS1 or R46A virus (see section 3.2.1) at an MOI of 1.5. 16 h p.i. cells were lysed with 1 ml GFP-Trap-lysis buffer (see section 3.4.1) and 100 l of cell lysate were mixed with 6x SDS sample buffer as WCL control. Remaining cell lysate was incubated with at least 20 l GFP-Trap®slurry rotating for 1 h at 4 C. GFP-Trap®beads were washed three times with GFP-dilution buffer by centrifugation (3 min, 2000 g) and bound proteins and interaction partners were eluted by incubation with 2x SDS sample buffer for 10 min at 95 C. Samples of WCL and precipitate were analysed by SDS PAGE and immunoblotting (see section 3.4.2, section 3.4.4).
3.4.7 Interferon ELISA
To measure IFN expression and to confirm the inhibitory effect of NF B inhibitor BAY 11-7085 (Enzo®Life Sciences) on IFN production, concentration of secreted IFN in the supernatants of infected cells was determined by enzyme-linked immunosorbent assay (ELISA).
Supernatants of treated cells were collected, cleared of debris by centrifugation and stored at 80 C. IFN levels were measured with the VeriKine™ Human IFN ELISA Kit (PBL) or with the Fujirebio®Inc. IFN ELISA Kit according to the manufacturers’ instructions.