Goat anti lamin b

Total cell lysates were prepared in lysis buffer [150 mM NaCl, 1% NP-40, 50 mM Tris, 0.2% sodium dodecyl sulfate (SDS), 5 mM NaF] containing a cocktail of protease inhibitors and phosphatase inhibitors (Roche). The following antibodies were used for Western blotting to detect cellular proteins: rabbit anti-Regnase-1 (Novus Biologicals), rabbit anti-SOX9 (Abcam), mouse anti-HIF-2α (Santa Cruz Biotech), and goat anti-Lamin B (Santa Cruz Biotech). For detection of secreted proteins (MMP3 and MMP13), 900 μl of serum-free conditioned medium was subjected to trichloroacetic acid precipitation, and the proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using rabbit anti-MMP3 (Abcam) and rabbit anti-MMP13 (Aviva Systems Biology) antibodies.

Whole cell lysates were prepared by boiling cell pellets for 10 min in SDS lysis buffer [2% SDS, 10% Glycerol, 62 mM Tris-HCl, pH 6.8 and a complete mini-protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. After protein quantification with Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA), equal amounts of proteins were loaded, separated on a polyacrylamide gel, and transferred to a nitrocellulose membrane. Protein bands were immuno-detected with appropriate antibodies, e.g., rabbit-anti-XPC [47 (link)], mouse-anti-E-Cadherin (BD Transduction Laboratories, San Jose, CA, Cat. No. 610181, 1:1000), rabbit-anti-Snail (Cell Signaling, Danvers, MA, Cat. No. 3879, 1:1000), rabbit-anti-pERK1/2 (Cell Signaling, Cat. No. 9101, 1:1000), rabbit-anti-ERK2 (Cell Signaling, Cat. No. 9108, 1:1000), mouse-anti-γH2AX (Millipore, Billerica, MA, Cat. No. 05-636, 1:1000), rabbit-anti-H2AX (Cell Signaling, Cat. No. 7631, 1:1000), mouse-anti-Actin (Santa Cruz Biotechnology, Dallas, TX, Cat. No. sc-47778, 1:1000), and goat-anti-Lamin B (Santa Cruz Biotechnology, Cat. No. sc-6216, 1:1000).

LifeAct-GFP-NLS-expressing HFFs (2 × 10⁵/well) were seeded in a 12-well plate and either mock infected or infected with WT HCMV (MOI of 1). At 72 hpi, cells were separated into nuclear and cytoplasmic fractions using an NE-PER extraction kit (Thermo). For Western blotting, each fraction was mixed with an equal volume of 2× Laemmli buffer (Bio-Rad), boiled at 95°C for 5 min, and run on a 4 to 20% SDS-polyacrylamide gel (Bio-Rad). Proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane, blocked with 5% milk in DPBS-T (DPBS with 0.5% Tween 20), and probed with primary antibodies overnight at 4°C with rocking. The following antibody dilutions were used: mouse anti-β-actin (Sigma; A5441), 1:5,000; rabbit anti-GFP (Invitrogen; A11122), 1:1,000; goat anti-lamin B (Santa Cruz; 6216), 1:200; rabbit anti-GAPDH (Cell Signaling; 14C10), 1:1,000. Membranes were washed 3 times with DPBS-T for 10 min at room temperature (RT) with rocking. Membranes were then incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) (Southern Biotech) at 1:1,000 for 1 h at RT with rocking, followed by washing. Finally, chemiluminescence solution (Pierce) was added to membranes and signal was detected with film.