Mouse monoclonal antibody to β actin

Cells were collected and lysed in lysis buffer (Thermo Fisher Scientific) after stimulation of mIL-11 (R&D systems), electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membranes (Biorad). After blocking, the membranes were incubated with primary antibodies overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 1 h. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher). The sources for the antibodies were as follows: Rabbit monoclonal antibodies to STAT1, STAT3, p-STAT1, HDAC4, HDAC5, GAPDH (dilution 1:1000). Rabbit polyclonal antibody to p-STAT3 (1:1,000), mouse monoclonal antibody to β-actin (1:1000), goat anti-rabbit IgG HRP-linked antibody, and goat anti-mouse IgG HRP-linked antibody (dilution 1:10,000) are from Cell Signaling (Danvers, MA). Mouse monoclonal antibody to NMP84 (dilution 1:1000) as a nuclear marker was obtained from Abcam, and STAT3-IN-1 from Selleck Biotech Co., Tokyo, Japan.

CPT1A protein expression was assessed 5 days following shRNA lentiviral infection. Cells were lysed in RIPA-2 (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) containing protease (Mini Complete; Roche) and phosphatase inhibitors (PhosSTOP; Roche). Equal amounts of lysate were separated by SDS-PAGE and transferred electrophoretically to a PVDF membrane (Millipore). Membranes were incubated in 5% nonfat milk in TBS (20 mM Tris, 135 mM NaCl) containing 0.1% Tween-20 (TBS-T) for 1 hr. After blocking, the membranes were probed with a 1:500 dilution of anti-CPT1A (Cell Signaling) in 0.5% nonfat milk in TBS-T. After a series of washes in TBS-T, the membranes were incubated with followed by a 1:10,000 dilution of HRP-conjugated goat anti-rabbit IgG (Cell Signaling). Antibody binding was detected using West Femto SuperSignal chemiluminescent reagents (ThermoScientific). Relative protein loading was determined with a 1:2,000 dilution of mouse monoclonal antibody to β-Actin (Cell Signaling) followed by a 1:5,000 dilution of HRP-conjugated sheep anti-mouse IgG (Amersham).