Fluorchemq multiimage 3

For in extract ubiquitination: (His)₆-p27 was bound to Ni-NTA beads (Qiagen) for 1 h, washed with 20 mM Tris pH 7.5 10 mM imidazole, beads were blocked with BSA for 1 h, and washed as before. Reactions were assembled containing 5× Buffer (200 mM Tris pH 7.6, 25 mM MgCl₂, 10 mM DTT), MG132, ATP, creatine phosphokinase, creatine phosphate, ubiquitin, yeast extract, and UbcH7. Reactions were stopped after 90 min by the addition of Laemmli sample buffer and run on 10.5% SDS-PAGE gels. Antibodies used were p27 (AF2256 R&D Systems), ubiquitin (made in house), blots were developed with ECL (Prometheus ProSignal Femto, Genesee Scientific) and imaged using a FluorChemQ MultiImage III (Alpha Innotech).
For reconstituted in vitro ubiquitination: reactions were assembled in buffer (final concentration: 40 mM Tris pH7.6, 5 mM MgCl2, 2 mM DTT) with ATP, creatine phosphokinase, creatine phosphate, ubiquitin, Ube1, UbcH7, p27, and SMURF1. Reactions were stopped after 90 min by the addition of Laemmli sample buffer and run on 10.5% SDS-PAGE gels. Antibodies used were SMURF1 (sc-100616 Santa Cruz Biotechnology), p27 (AF2256 R&D Systems), ubiquitin (made in house), blots were developed with ECL (Prometheus ProSignal Femto Genesee Scientific) and imaged using a FluorChemQ MultiImage III (Alpha Innotech).

Expression of viral and host cellular proteins was detected by Western blotting according to the procedure described previously (10 (link)). Equal amounts (10 to 30 μg) of protein samples were separated using 10 to 12% SDS-PAGE gels and transferred to 0.2-μm nitrocellulose membranes. Anti-PSMA1 (Invitrogen; catalog no. PA1-963), anti-PSMA2 (Cell Signaling; catalog no. 2455), anti-PSMA6 (Invitrogen; catalog no. PA576058), anti-beta-actin (Cell Signaling; catalog no. 3700S), anti-STAT3 (Cell Signaling; catalog no. 9139S), anti-CST3 (Cell Signaling; catalog no. 4280), and in-house-prepared IAV mouse-anti-NS1 and mouse-anti-NP (93 (link)) were used to detect specific proteins. Appropriate secondary horseradish peroxidase (HRP)-conjugated horse anti-mouse or anti-rabbit (Cell Signaling; catalog no. 7076 and 7074, respectively) were used to detect immune complexes. Protein bands were developed with ECL reagents and captured by Alpha Innotech FluorChemQ MultiImage III. The differences in protein expression were determined by measuring band intensities with ImageJ 1.50i (NIH, USA). The data were analyzed and graphically presented by GraphPad Prism v 9.1.0 software.

PFGE was performed as previously described [25 (link)] with modifications enabling greater separation of high-molecular-weight DNA molecules, such as chromosome XII, from the rDNA cloud. Plugs were placed in the wells of a 0.8% agarose gel (D5 agarose (Conda, Torrejon de Ardoz, Madrid, Spain) in 1x TAE) and sealed with the same agarose. Until stated otherwise, electrophoresis was performed for 24 hours in 1x TAE buffer at 6 V/cm, 12°C, ramping 0.8, angle 120°, switch time 60–85 s, using a CHEF Mapper® XA Pulsed Field Electrophoresis System (BioRad, Hercules, CA, USA). After electrophoresis, DNA was stained with 0.5 μg/ml ethidium bromide (Sigma-Aldrich) for 30 min, washed twice with water for 15 min, and photographed using a 302-nm UV light for DNA visualization with a charge-coupled device camera (Fluorchem Q Multi Image III, Alpha Innotech, San Leandro, CA, USA).