Adipose tissues were homogenized in ice-cold lysis buffer. After homogenization for 30 min on ice, the tissue homogenates were centrifuged at 13,000 r/min for 30 min at 4 °C. The bicinchoninic acid method was applied to determine the total protein concentration. Equivalent amounts of proteins were loaded in each lane, denatured, and then frozen at − 80 °C before western blotting. The proteins were subsequently subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After incubation with the apposite primary antibody at 4 °C overnight and secondary antibodies at room temperature for 2 h, polyvinylidene fluoride membranes were washed 3 times in PBS for 10 min, and the targeted proteins were detected by using enhanced chemifluorescence reagent.
The membranes were incubated with antibodies against TLR4 (ab13556, Abcam), IRS-1 (ab131487, Abcam), MyD88 (ab2064, Abcam), NF-κB (8242S, CST), andphospho-NF-κB (3033, CST). All the samples were analysed in an average of 3 independent tests by using different gels. Bands were quantified by ImageJ.
The membranes were incubated with antibodies against TLR4 (ab13556, Abcam), IRS-1 (ab131487, Abcam), MyD88 (ab2064, Abcam), NF-κB (8242S, CST), andphospho-NF-κB (3033, CST). All the samples were analysed in an average of 3 independent tests by using different gels. Bands were quantified by ImageJ.