The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered.
Protein quantity was assessed (Dc Protein Assay; Bio-Rad) and proteins were separated by SDS-PAGE (4 10% gradient gel), transferred to a nitrocellulose membrane and probed with antibodies against AMPKα (Cell Signaling), phospho-AMPKα (Thr172) (Cell Signaling), PPARG (Santa Cruz), dp-ERK (Sigma), phospho-STAT3 (Tyr705) (MBL) and/or actin (Sigma).
Proteins were separated on SDS-PAGE gradient gels, transferred to a PVDF membrane and probed for proteins of interests and secondary HRP-conjugated antibodies were used for detection.
Aliquots (15 μg) of samples were separated on 5 20% polyacrylamide gradient gels, transferred to a polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA, USA), and hybridized with primary antibodies diluted in Tris-buffered saline containing 6% skim milk and 0.05%Tween-20Tween-206% milk/TBST) overnight at 4°C.
The dried lipid extracts were dissolved in 1.0 ml of chloroform (gradient grade) and transferred to a transmission flow cell (path length: 0.025 mm) with sodium chloride crystals.
An aliquot of the lysate (25 μg protein) was boiled in SDS sample buffer, resolved on a 4 12% SDS-PAGE gradient gel, and transferred to a 0.2 μm nitrocellulose membrane.
Whole-cell extracts (NIH 3T3, 35 50 µg protein; MEFs, 125 250 µg) were resolved on a 4 20% gradient SDS-PAGE and transferred to a nitrocellulose membrane (BioRad).
Equal amounts of protein were separated by SDS-PAGE (4 12% gradient gels), and proteins were transferred to a nitrocellulose membrane (Invitrogen).
The samples were then separated using a NuPage 4 12% gradient polyacrylamide gel (Invitrogen) and transferred to a PVDF membrane.
Then, 30 μl of each extract was run on a 4 to 20% gradient SDS-polyacrylamide gel and transferred to a polyvinyl difluoride (PVDF) membrane.
