Benzamidin

For subcellular fractionation, cells were washed twice with cold DPBS and re-suspended in lysis buffer (12.5 mL 1 M HEPES, ph 7.5, 7.5 mL 5 M NaCl, 1.25 mL 200 mM EGTA, 25 mL 100% Gycerin, 2.5 mL Triton X-100, 1.05 g NaF, 1.11 g Na₄P₂O₇ × 10 H₂O) containing the protease inhibitors Orthovandat (Sigma aldrich), Leupeptin (Sigma aldrich), Benzamidin (Sigma aldrich), PMSF (Sigma aldrich), Aprotinin (Sigma aldrich). After sonification, cells were centrifuged at 13.000 rpm for 5 min, and supernatants were transferred to new cups and incubated on ice.
For co-immunoprecipitation, 500 μg of the lysates was immunoprecipitated with 4 µL of the indicated antibodies and protein G or A agarose (Roche Diagnostics). The immunoprecipitates were subjected to immunoblotting.
For Western blotting, protein extracts were analyzed by SDS–PAGE and blotted onto nitrocellulose. Upon protein extraction and gel transfer, membranes were washed in TBS washing buffer and incubated with peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized by means of an enhanced chemiluminescence detection system (Western blotting detection reagent, GE healthcare). Membranes were probed with anti-NFATc2 (Santa Cruz), anti-Sp1 (Santa cruz) and anti-MEF 2A (Upstate cell signaling solutions). Anti-Lamin B (Santa cruz) and anti-ß-actin (Sigma-Aldrich) antibodies were used as loading control.

Proteins were extracted after tissue lysis with NP- 40 buffer (20 mM Tris HCl pH 8.0, 135 mM NaCl, 1% NP-40 and 10% glycerol) containing protease and phosphatase inhibitors (1 mM PMSF, 0.45 mg/ml benzamidin, 1 mM leupeptin, 1 mM aprotinin and 5 mM sodium orthovanadate) (Sigma Aldrich, St. Louis, MO, USA). Concentration was determined^{64 (link)} for each animal, and thirty µg of proteins were separated into 12% SDS-PAGE, transferred onto nitrocellulose membranes (GE Healthcare, Wauwatosa, Wisconsin, USA), and blocked. The following antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used: rabbit anti- GR (5 µg/ml) (M20); goat anti- pepsinogen C (0.1 µg/ml) (I-19), monoclonal anti- Mist1 (5 μg/ml) (6E8), monoclonal anti- Moesin (5 μg/ml) (38/87) overnight at 4 °C. Monoclonal anti- β-actin (1 µg/ml) (Sigma Aldrich) was used as internal loading control. Reactions were developed with ECL Kit (GE Healthcare) and detected in X-ray films (Kodak, MXG-Plus, Rochester, NY, USA) (Supplementary Fig. 1). Densitometry was performed with ImageJ (1.37 v Software, NIH Public Domain) and bands were cropped to represent control and NMS groups. Gels were run in replicates to confirm results.

Cellular and viral proteins were analyzed by Western blot. After a wash step with PBS, cells were lysed in RIPA (radioimmunoprecipitation) buffer (137 mM NaCl (Roth), 25 mM Tris–HCl (pH 8) (Roth), 2 mM EDTA (pH 8) (Roth), 10% (v/v) glycerol (Roth), 1% (v/v) NP-40 (Sigma-Aldrich), 0.5% (w/v) sodium deoxycholate (Serva), 0.1% (w/v) SDS (Roth)) supplemented with protease inhibitors (1:1000 Pefablock (200 mM) (Roth), 1:1000 Leupeptin (5 mg/ml) (Serva), 1:1000 Aprotinin (5 mg/ml) (Roth), 1:100 Na₃VO₄ (100 mM) (Sigma), and 1:200 Benzamidin (1 M) (Sigma)) centrifuged at 4 °C and 20,000 rpm for 10 min. Cleared lysates were mixed with 5 × Laemmli buffer and headed for 2 min at 95 °C. Protein separation was performed by SDS-PAGE and protein transfer onto nitrocellulose membranes by Western blotting, followed by blocking in TBS-T buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 0.2% Triton X-100 (Roth), 1% Tween-20 (Roth)) containing 3% BSA (w/v) (Roth) for 1 h. Membranes were incubated for 1 h or overnight with primary antibodies (Table S1) diluted in blocking buffer. A 45 min incubation with 1:3000 secondary antibody (Table S1) dilutions in TBS-T was performed to detect the primary antibodies using chemiluminescence and the Li-CorOdissey^® Fc Imaging System. Signals were analyzed by the Image Studio™ (LiCor) software.