Mouse anti rabbit igg hrp

20 μg of cells or 40 μg of tissue lysates were subjected to electrophoresis on 10% acrylamide gels and transferred to PVDF membranes. The membranes were incubated for 1 h with blocking buffer (either TBS-T containing 5% [w/v] BSA or 5% skim milk). The membranes were then incubated with the indicated primary antibodies diluted in blocking buffer (1:1,000) for 12 h at 4 °C: anti-pAmpk-α Thr172 (Cell Signaling, #2535s), anti-Ampk-α1 (R&D, #AF3197), anti-Ampk-α (Cell Signaling, #2532), anti-Ampk-α2 (abcam, ab3760), anti-UCP1 (abcam, ab10983), anti-HSP90 (Cell Signaling, #4874), anti-Bcl2 (abcam, ab196495), anti-cleaved Caspase 3 (Cell Signaling, #9661), anti-GAPDH (Cell Signaling, #2118), anti-Reg3γ (abcam, ab198216), and anti-Reg3α (abcam, ab202057). The membranes were washed three times with TBS-T and incubated with the secondary HRP-conjugated antibodies mouse anti-rabbit IgG-HRP (Cell Signaling, #7074S) (diluted 1:2000 in 5% skim milk) at room temperature for 1 h. Finally, the membranes were washed in TBS-T three times for 10 min each, and the signal was detected using enhanced chemiluminescence reagent (Pierce, IL, USA). Protein levels were quantified by densitometry using Image J software.

Protein was extracted from mouse livers. Total protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. These membranes were then blocked and incubated with primary antibodies against ApoA5 (Abcam, Cambridge, MA, United States, ab239579), TLR4, MyD88, NF-κBp65, and β-actin (Cell Signaling Technology, Beverly, MA, United States, #14358, #4283, #8242 and #4970). Membranes were incubated for 1 h with the following secondary antibodies: goat anti-mouse IgG-HRP and mouse anti-rabbit IgG-HRP (Cell Signaling Technology, Beverly, MA, United States, #43593 and #58802). Signal detection was performed using SuperSignal Dura Substrate (Pierce, Biotechnology, United States), after which immunoblot signals were quantified using Quantity One software.