Proteinase inhibitors and phosphatase inhibitors

One million J-Lat A1 cells or PBMCs from HIV-negative uninfected controls were incubated with 12 nM PEP005 for 6 hrs. Whole cell protein extracts were prepared with RIPA buffer containing proteinase inhibitors and phosphatase inhibitors (Sigma). Expression of the isoforms of PKC protein or NF-κB/p65 was evaluated using the PKC Isoform Sampler Antibody Kit (Cell Signaling, 9960S) or anti-NF-κB/p65 (Abcam). The level of phosphorylation of PKC was determined using anti-Phospho-Ser664-PKCδ, anti-Phospho-Ser643/676 PKCδ or anti-Phospho-T538-PKCθ (Millipore), and the level of phosphorylation of IκB was determined using p-IκBα (Ser32), p-IκBβ (Thr19/Ser23) or p-IκBε (Ser18/22) antibodies (Millipore).

Cultured CT26.WT cells, tumors or liver tissues lysed in RIPA buffer with PMSF, proteinase inhibitors and phosphatase inhibitors (Sigma-Aldrich) to yield the homogenate. Following the total protein levels were quantified via the BCA protein assay kit (Thermo Fisher Scientific, USA). Equivalent amounts of 40 µg protein extracts were loaded and electrophoresed with 10% SDS-PAGE, and then transferred unto the polyvinylidene fluoride (PVDF) membranes. Next, after blocking with 3% BSA, the membranes were incubated overnight with the corresponding primary antibodies, subsequently washing and incubating with secondary antibodies (goat anti-rabbit). Finally, immunoreactivity was scanned with an enhanced chemiluminescence system. All experiments were conducted out in triplicate independently.

Primary culture of SMCs was treated with 100 ng/ml recombinant PDGF-BB (R&D Systems) or 100 ng/ml AREG (R&D Systems) for 72 h. Whole cell lysate was then prepared by a RIPA buffer (Sigma Aldrich, St. Louis, MO) supplemented with proteinase inhibitors and phosphatase inhibitors (Roche). Protein concentration was determined by a bicinchoninic acid (BCA) method (Pierce BCA Protein Assay Kit, Thermo Scientific). After Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE), separated proteins were transferred to a PVDF membrane (Hybond-P, GE healthcare, Buckinghamshire, UK) and blocked with an ECL plus blocking agent (GE healthcare). Membranes were then incubated with primary antibodies followed by incubation with anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by a chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
The following primary antibodies were used: mouse monoclonal anti-SMA antibody (#M0851, Dako) and mouse monoclonal anti-α-tubulin antibody (#T6199, Sigma Aldrich).