Anti nf κb antibody

Cells were collected and lysed in ice-cold RIPA lysis buffer. Sixty micrograms of protein was electrophoresed by SDS-PAGE using 10% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in phosphate-buffered saline with 0.05% Tween 20 for 1 h. The membranes were probed using anti-NF-κB antibody (1:200; Santa Cruz, Shanghai, CN), anti-CREB antibody (1:500; GeneTex, CA, USA), anti-HIF-1α antibody (1:500; GeneTex, CA, USA) or anti-β-actin antibody (GeneTex, CA, USA) followed by anti-mouse or anti-rabbit secondary antibodies (1:10,000; GeneTex, CA, USA). The protein bands were developed using the ChemiLucent ECL Detection System (Millipore, MA) and were visualized using the Biospectrum AC Imaging System (UVP, CA, USA).

BEAS-2B cells and MLE-12 cells were cultured on sterile coverslips in 12-well plates overnight. The cells were fix with 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 30 min at room temperature. Then, the cells were blocked with normal goat serum (ORIGENE, SP-9000, Beijing, China) for 30 min at room temperature and incubated with anti-NF-κB antibody (SANTA CRUZ, Dallas, TX, USA, #SC-8008) overnight at 4 °C. The cells were washed 3 times with PBS and then incubated with goat anti-rabbit IgG Alexa Fluor^® 647 (Abcam, ab150115, Cambridge, UK) in the dark for 1 h at 37 °C. A fluorescent sealing liquid solution, containing DAPI was added to mark nuclei. The images were observed and collected using a fluorescence microscope (Invitrogen EVOS™ FL Auto 2 Imaging System, AMAFD2000, Thermofisher, Waltham, MA, USA).

Immunohistochemistry for myostatin and NF-κB was performed according to a previously described protocol [26 (link)-29 ]. In brief, 4 μm thick sections of paraffin-embedded gastrocnemius skeletal muscle were prepared, deparaffinized, and rehydrated. Next, 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. Phosphate-buffered saline (PBS, pH 7.2) was used to wash the sections before and after incubating some of them with myostatin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubating others with anti-NF-κB antibody (Santa Cruz Biotechnology), using the dilutions suggested by the manufacturers. The sections were then incubated with a biotinylated secondary antibody (LSAB kit, Dako, Carpinteria, CA, USA) and washed with PBS before being successively incubated with streptavidin horseradish peroxidase (LSAB kit, Dako). They were then rinsed with PBS and treated with 3,3′-diaminobenzidine substrate (Santa Cruz Biotechnology) until the desired color intensity was detected. Finally, the sections were washed with tap water to stop the further reaction. Hematoxylin (Santa Cruz Biotechnology) was used to counterstain the sections. Negative control slides were processed in the absence of a primary antibody. The sections were viewed at 40x under a light microscope (BEL Engineering Company, Italy).