Protean tgx gel

Heart tissue samples were isolated from 7-week- and 6-month-old mice after being euthanized using CO₂ and homogenized using a Cryolys-cooled Precellys 24 bead homogenizer (Bertin Corp.) using a combination of 1.4 mm and 2.8 mm ceramic beads at 6000 rpm for three bouts of 15 s in homogenization buffer (0.025 M Tris–HCl, 0.15 M NaCl, 0.001 M EDTA, 1% v/v NP-40, 5% v/v glycerol, pH=7.4), as previously described (35 (link)). Homogenates were then centrifuged for 30 min at high speed at 4 °C. Following quantification by bicinchoninic acid assay (Pierce), 30 to 40 μg of lysates were separated on 4 to 15% precast ProteanTGX gels (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were blocked at room temperature (RT) for 1 h and then incubated in primary antibody overnight at 4 °C. Primary antibodies targeted AnkB (1:1000, Covance antibody) (7 (link)), NCX1 (1:1000, Cell Signaling Technology CST 79350), NKA (1:1000, Cell Signaling Technology CST 3010), and Ankyrin-G (1:1000, Covance, a gift from Dr Paul Jenkins) (36 (link)). Secondary antibodies used were donkey anti-rabbit or donkey anti-mouse (Jackson ImmunoResearch Laboratories, Bio-Rad). Densitometry analysis was performed using ImageJ software (https://imagej.net/ij/index.html).

Cells were lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA) with protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA). Protein extracts were clarified by centrifugation, resolved by SDS-PAGE using PROTEAN® TGX™ gels (Bio-Rad Laboratories, Hercules, CA), and transferred to PVDF membranes. Antibodies used in the study are listed in Supplemental Methods.

CCL2 HeLa cells were incubated at 4°C for 1 h, adsorbed for 1 h at 4°C with T1L or T3D at an MOI of 1×10⁴ particles per cell. Cells were washed with cold PBS, pre-warmed complete media was added to cells, and whole-cell lysates were prepared using RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.1% sodium deoxycholate) that was supplemented with Protease Inhibitor Cocktail (Sigma), Phosphatase Inhibitor Cocktail (Sigma), sodium vanadate (Cell Signaling Technologies), and phenylmethylsulfonyl fluoride (PMSF, Sigma). Whole-cell lysates were resolved by gel electrophoresis using PROTEAN TGX-gels (Bio-rad) and immunoblotted using reovirus-specific polyclonal antiserum (1:1000) and actin (1:1000). Immunoblots were scanned using a LI-COR Odyssey infrared imaging system. The intensity of immunoblot bands was quantified using Image Studio software (LI-COR) and the ratio of the intensity of δ protein to that of total μ1 protein was calculated.