PMC:7432599 / 47954-50103
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7432599","sourcedb":"PMC","sourceid":"7432599","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7432599","text":"4.14. Immunoblotting\nTissues were homogenized at 4 °C in ice-cold RIPA buffer containing phosphate-buffered saline (PBS), 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM sodium fluoride, 2 mM sodium orthovanadate, and 1 tablet/10 mL of protease inhibitor mixture (Complete Mini, Roche Diagnostics) in a TissueLyser (Qiagen). The samples were incubated on ice for 30 min, centrifuged at 17,400× g for 15 min at 4 °C, and the supernatant was transferred to a new tube and stored at −80 °C until use. Protein concentration was determined with the Lowry method. Protein samples (40 µg) were separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membranes (Hybond-P, Amersham, GE Healthcare, Chicago, IL, USA) using a wet electroblotting System (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h with 5% non-fat dry milk, and incubated with primary antibody diluted in blocking solution overnight. Primary antibodies were as follows: AMPKa 1/2 (Santa Cruz Biotechnology, dilution 1:3000 liver and 1:1500 skeletal muscle), p-AMPK, Th2-172 (Santa Cruz Biotechnology, dilution 1:1000 liver and skeletal muscle), JNK (Merck Millipore, Burlington, MA, USA) dilution 1:1000 liver), p-JNK (Thr183/Tyr185, Thr221/Tyr223) (Cell Millipore dilution 1:1000 liver), UCP-1 (abcam dilution 1:1000 brown adipose tissue), GAPDH (Abcam 1:100 brow adipose tissue), HSL (Cell Signaling Danvers, MA, USA, dilution 1:2000 white adipose tissue), p-HSL (Cell Signaling dilution 1:2000 white adipose tissue). The membranes were washed three times with TBS-T for 10 min and then incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit or rabbit anti-goat 1:3500) for 1.5 h. Visualization was performed using a chemiluminescent detection reagent (Millipore, MA, USA). Digital images of the membranes were obtained by a ChemiDoc MP densitometer and processed by Image Lab software (Bio-Rad). The results are reported as phosphorylated/total protein ratio. A value of 1 was arbitrarily assigned to the control group, which were used as a reference for the other conditions.","divisions":[{"label":"title","span":{"begin":0,"end":20}}],"tracks":[]}