Novex tris glycine sds sample buffer 2

Immunoblotting was performed as described previously.²⁴ Briefly, CD4 + T cells (or CD19 + B cells for the indicated immunoblots) were isolated from splenocytes using magnetic or flow‐cytometric cell sorting and lysed in 2 × Lämmli Buffer (Novex Tris‐Glycine SDS Sample Buffer 2×; Thermo Fisher Scientific Waltham, MA, USA) supplemented with 100 mM DTT (Sigma Aldrich, St. Louis, MO, USA), protease inhibitors (cOmplete ULTRA Tablets; Roche, Basel, Switzerland), and phosphatase inhibitors (PhosSTOP; Roche) for 5 minutes at 99°C. Primary antibodies to phospho‐AMPKα1 (p‐AMPKα1, Thr172), total AMPKα1, phospho‐acetyl‐CoA carboxylase (p‐ACC, Ser79), total ACC, phospho‐TSC2 (p‐TSC2, Thr1462, and Ser1387), total TSC2, phospho‐Raptor (p‐Raptor, Ser792), total Raptor, GAPDH, and pan‐Actin were obtained from Cell Signaling Technology (Cell Signaling Technology, Cambridge, UK). All primary antibodies were used at a dilution of 1:1000. HRP‐conjugated goat anti‐rabbit IgG (Dako, Agilent Technologies, Santa Clara, CA, USA) was used as the secondary antibody at a dilution of 1:2000. Immunoblots were developed using a HRP‐specific substrate (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific). HRP chemiluminescence signals were detected using a Fujifilm LAS‐4000 image analyzer (GE Healthcare, Chicago, IL, USA) and analyzed using Fiji (ImageJ, https://imagej.net/Fiji).

The concentrated NDV-HXP-S or WT NDV was mixed with Novex™ Tris-Glycine SDS Sample Buffer (2×) (Thermo Fisher Scientific), NuPAGE^TM Sample Reducing Agent (10×) (Thermo Fisher Scientific), and PBS at appropriate amounts to reach a total protein content of 20 µg in 50 µl volume. The mixture was heated at 90 °C for 5 min. The samples were mixed by pipetting and loaded at 30 µg to a 4–20% 10-well Mini-PROTEAN TGX^TM precast gel (Bio-Rad). Ten microliters of the Novex^TM Sharp Pre-stained Protein standard (Thermo Fisher Scientific) were used as the ladder. The electrophoresis was run in Tris/Glycine SDS/Buffer (Bio-Rad). The gel was then washed with distilled water at room temperature several times until the dye front in the gel was no longer visible. The gel was stained with 20 mL of SimplyBlue^TM SafeStain (Thermo Fisher Scientific) for a minimal of 1 h to overnight. The SimplyBlue^TM SafeStain was decanted and the gel was washed with distilled water several times until the background was clear. Gels were imaged using the Bio-Rad Universal Hood Ii Molecular imager (Bio-Rad) and processed by Image Lab Software (Bio-Rad).

To generate sucrose-gradient, 12.5 mL of 10% (w/v) sucrose in 0.0125 M citrate buffer was laid on top of 12.5 mL 60% (w/v) sucrose in 0.0125 M citrate buffer in round-bottom polypropylene copolymer centrifuge tubes (Thermo Fisher Scientific). The vertical tubes were then sealed using parafilm and gently flipped to a horizontal position to allow diffusion to occur at RT for 1:30 h. The tubes were then returned to a vertical position and kept in the fridge at 4 °C overnight to ensure sufficient diffusion. Five hundred microliters of the concentrated virus obtained from sucrose-cushion purification described above were laid on top of the gradient and ultracentrifugation was performed at 25,000 rpm for 2 h at 4 °C in a Beckman L7-65 ultracentrifuge. One mL of each fraction was collected with a total of 26 fractions (the last fraction is <1 mL). Twenty microliters of each fraction were mixed with 25 µl of Novex™ Tris-Glycine SDS Sample Buffer (2×) (Thermo Fisher Scientific) and 5 µl of NuPAGE^TM Sample Reducing Agent (10×) (Thermo Fisher Scientific) to reach a total volume of 50 µl. The mixture was treated at 90 °C for 5 min. Thirty microliters of each sample were resolved on 4–20% of SDS–PAGE. The electrophoresis and the protein staining were performed as described above.