Lamin B1 Monoclonal Antibody (L-5)

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	-Mouse (1:1000)

Protocol tips
-Mouse (1:1000)

Publication protocol

Overnight subconfluent cultures of Ohio-HeLa cells with or without infection with HRV16 at an MOI of 1 were collected for whole-cell lysates or cytoplasmic/nuclear protein extraction at the specified time points. Cytoplasmic and nuclear protein extractions were performed using the NE-PER kit according to the manufacturer's recommendations (ThermoFisher). Whole-cell lysates were collected at the specified time points in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris, pH 8) with protease and phosphatase inhibitors (cOmplete, PhosSTOP; Roche) added. Protein extracts were heated at 100°C for 5 min in Laemmli buffer (63 mM Tris-HCl, pH 6.8, 0.1% 2-mercaptoethanol, 0.0005% bromophenol blue, 10% glycerol, 2% SDS) prior to SDS-PAGE. Whole-cell lysates and subcellular fractions were subjected to SDS-PAGE using 10% or 12.5% polyacrylamide gels, followed by protein transfer to nitrocellulose membranes in Tris-glycine-ethanol buffer (25 mM Tris-HCl, 192 mM glycine, 20% ethanol) for 90 min at 400 mA. The blots were stained with Ponceau S (Sigma) to confirm transfer and then blocked for 1 h in 4% skim milk (Diploma) in phosphate-buffered saline (PBS) (10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.2, 2.7 mM KCl, 137 mM NaCl) prior to incubation with primary antibodies diluted in 1% skim milk in PBS-T (PBS containing 0.1% Tween 20) overnight at 4°C with rocking. After the blots were washed in PBS-T, they were incubated with species-specific secondary antibodies conjugated to horseradish peroxidase diluted at 1:5,000 in 1% skim milk in PBS-T, followed by washing and detection of bound antibodies with enhanced chemiluminescence (ECL; PerkinElmer). Protein bands were detected using the Licor Odyssey Fc, and captured digital images were analyzed using ImageStudio software (Licor). Where required, blots were stripped to remove bound antibodies (2% SDS, 62.5 mM Tris-HCl, pH 6.8, 114.4 mM β-mercaptoethanol) at 50°C for 10 min, followed by washing in PBS-T, blocking in 4% skim milk in PBS, and reprobing using different primary antibodies.

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Manufacturer protocol

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