Ab12135

Proteins were prepared from liver tissues in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors cocktail (Roche, Switzerland). Then SDS-PAGE was conducted, and bands were transferred to PVDF membrane, incubating with primary antibodies against toll-like receptor 4 (TLR4, diluted at 1 : 1000, catalog number: ab45104, Abcam, Cambridge, UK), microtubule-associated protein light chain 3 (LC3, diluted at 1 : 1000, catalog number: ab48394, Abcam, Cambridge, UK), p62 (diluted at 1 : 1000, catalog number: ab56416, Abcam, Cambridge, UK), phosphor-IκB (diluted at 1 : 1000, catalog number: ab12135, Abcam, Cambridge, UK), IκB (diluted at 1 : 1000, catalog number: 4814, Cell Signaling Technology, Boston, USA), caspase-3, caspase-8, caspase-9, and poly-(ADP-ribose) polymerase (PARP) (diluted at 1 : 1000, catalog numbers: 9665, 4790, 9508, and 9532, Cell Signaling Technology, Boston, USA). Corresponding secondary antibodies (diluted at 1 : 5000, catalog numbers: ZDR-5306 or ZDR-5307, Sino Biological, Beijing, China) were added, and bands were visualized with enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, USA). Signals were normalized to that of β-actin (diluted at 1 : 5000, catalog number: 4967, Cell Signaling Technology, Boston, USA). Five replicates were used for statistical analysis.

Proteins were prepared from liver tissues in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors cocktail (Roche, Switzerland). Then SDS-PAGE was conducted and bands were transferred to PVDF membrane, incubating with primary antibodies against toll-like receptor 4 (TLR4), microtubule-associated protein light chain 3 (LC3), phosphor-IκB and phosphor-ACC (diluted at 1:1000, catalog number: ab45104, ab48394, ab12135 and ab68191, respectively, Abcam, Cambridge, UK), phosphor-NFκB, NFκB, IκB, caspase-3, caspase-8, caspase-9, poly-(ADP-ribose) polymerase (PARP), and phosphor-AMPK (diluted at 1:1000, catalog number: 3033, 8242, 4814, 9665, 4790, 9508, 9532, and 2535, respectively, Cell Signaling Technology, Boston, USA). Corresponding secondary antibodies (diluted at 1:5000, catalog numbers: ZDR-5306 or ZDR-5307, Sino Biological, Beijing, China) were added and bands were visualized with enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, USA). Signals were normalized to that of β-actin (diluted at 1:5000, catalog number: 4967, Cell Signaling Technology, Boston, USA).

The tissue samples were ground under liquid nitrogen and the cell samples were washed with Hank’s balanced salt. Then, the samples were lysed in ice-cold RIPA lysis buffer (Beyotime Inc., Nanjing, China) in the presence of protease inhibitors (Sigma, P8349) and incubated on ice for 40 min. For determination of NF-κB signal pathway, the cytoplasmic and nuclear proteins were extracted from the primary cortical microglial cells using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China). Total protein content was measured using a BCA protein assay kit (Applygen, China). Then the proteins were separated by SDS-PAGE using 10% gels and transferred onto PVDF membranes (Thermo Fisher Scientific, USA). After blocking for 30 min at room temp in blocking solution containing 5% non-fat milk, the membranes were incubated overnight at 4 °C with primary antibody: anti-Iba1 antibody (1:1000, ab178847, Abcam); NF-κB p65 (1:50,000, ab32536, Abcam), IκBα (1:1000, ab109300, Abcam) and pIκBα (1:1000, ab12135, Abcam). After several washes, the membranes were incubated with an appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The proteins bands were visualized using ECL kits (Amersham) and the optical density of the protein bands was quantified using the ImageJ software. Beta-actin was used as an internal control.