Anti nf κβ p65 monoclonal rabbit ab

Cell and tissue lysates (prepared as described above) were assayed by Precision Red Advanced Protein Reagent to assess total protein concentration. Equal quantities of protein were aliquoted, and samples were brought to equal volume in Laemmli buffer and water before boiling for 10 min at 100 °C. Samples were resolved in acrylamide gels and transferred to PVDF membranes with a 0.45-μm pore size (Milipore; Cat No. IPVH00010). PVDF membranes were blocked in 5% bovine serum albumin (BSA) for at least 1 h before overnight incubation with primary antibodies diluted in Tris-buffered saline with 0.1% tween (TBST). Primary antibodies used are as follows: anti-Cox2 polyclonal rabbit Ab (Abcam; Cat No. Ab52237; 1:1000), anti-NF-κΒ p65 monoclonal rabbit Ab (Cell Signaling; Cat No. D14E12; 1:2000), anti-Poldip2 monoclonal rabbit Ab (Abcam; Cat No. Ab181841; 1:2000), and anti-tubulin polyclonal rabbit Ab (Abcam; Cat No. Ab6046; 1:5000). Next, membranes were washed with TBST for 1 h before incubation with secondary antibody for an additional hour. Anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cell Signaling; Cat No. 7074S) was used at half the concentration of primary antibodies. Bands were visualized using enhanced chemiluminescence (ThermoFisher; Cat No. 34580) and detected with Amersham Hyperfilm ECL (GE).

RBMVECs were seeded and cultured on gelatin-coated glass coverslips until confluent. Confluent monolayers were then transfected with Poldip2 or control siRNA as described above. Forty-eight hours post-transfection, cells were stimulated with 1 μg/mL LPS for 1 h. After 1 h, cells were washed twice in HBSS and fixed in 3.7% paraformaldehyde diluted in HBSS for 10 min at room temperature. Cells were then washed three times in PBS and permeabilized in PBS containing 0.2% Triton X-100 for 5 min followed by three washes in PBS for 2 min each. Blocking was performed in 0.5% BSA in PBS for 30 min. Anti-NF-κΒ p65 monoclonal rabbit Ab (Cell Signaling; Cat No. D14E12) was diluted 1:500 in blocking buffer and coverslips were incubated overnight at 4 °C with gentle rocking. The following day, coverslips were rinsed in blocking buffer (three washes × 5 min each) before incubation with secondary (Alexa488) antibody (1:500 dilution, Invitrogen) and DAPI (1:1000 dilution) for 1 h. Cells were then rinsed three times in PBS for 5 min each. Finally, coverslips were mounted on glass slides using Fluoromount-G (SouthernBiotech; Cat No. 0100-01).