Hrp labeled anti rabbit igg

Thirty micrograms of protein from each lysate was boiled for 5 min in SDS sample buffer [24] (link). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon™-P, MerckMillipore, Billerica, MA, USA). Antibodies against adipose triglyceride lipase (ATGL) (MAB11192, R&D Systems™, MN, USA), peroxisome proliferator activated receptor γ (PPAR γ; H-100, Santa Cruz Biotechnology, Inc., TX, USA), fatty acid synthase (FAS; F9554, Merck, Darmstadt, Germany), β-actin (MAB1501, MerckMillipore), HRP-labeled anti-mouse IgG (Thermo Fisher Scientific Inc.), and HRP-labeled anti-rabbit IgG (Thermo Fisher Scientific Inc.) were also used for Western blotting. All immunoreacted proteins were detected using ECL™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK).

Lung tissues from mice and NCI-H460 human lung cells were homogenized with a Dounce homogenizer in lysis buffer at 4 °C. The homogenates were centrifuged at 15,000× g for 20 min at 4 °C, and the supernatants were transferred to different tubes, followed by measuring the protein concentrations. Samples (30 μg of protein) were subjected to 8–12% polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto nitrocellulose membranes, which were incubated with primary antibodies (Cleaved poly (ADP-ribose) polymerase (PARP) (Abcam, Cambridge, MA, USA); Cleaved caspase 3, p53 upregulated modulator of apoptosis (PUMA) (Cell Signaling Technology, Danvers, MA, USA); Actin, BAX, Cytochrome c, p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Prx-SO₃ (AbFrontier, Seoul, Korea)), followed by incubation with HRP-labeled anti-rabbit IgG (ThermoScientific, Waltham, MA, USA). Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK), and band intensities were quantified with ImageQuant 5.2 software (Molecular Dynamics Ltd., Cambridge, CB2 8PQ, UK).

Protein extracts were prepared from skin tissuess, HEM or B16F0 cells, resolved on SDS-polyacrylamide gels by electrophoresis, and blotted to PVDF membranes (Merck-Millipore, IPVH00010). After blocking in 5% non-fat milk (BD, 232100) dissolved in Tris-buffered saline (TBS) containing Tween 20 (Biosesang, TR2007-100-74), blots were incubated with primary antibody at 4°C for 15 h followed by secondary antibody at 25°C for 1 h. Immune complexes on the gels were detected using chemiluminescence kit (Thermo Fischer Scientific, 34095; GE Healthcare, RPN2232). Primary antibodies were anti-AMPKα (Cell Signaling Technology, 2532); anti-CREB (Cell Signaling Technology, 9197); anti-CRTC1 (Cell Signaling Technology, 2587); anti-CRTC2 (Merck Millipore, ST1099); anti-CRTC3 (Santa Cruz Biotechnology, sc-390712); anti-MITF-M (Abcam, ab12039); anti-p-AMPKα (Cell Signaling Technology, 4185); anti-p-CREB (Cell Signaling Technology, 9198); anti-p-CRTC1 (Cell Signaling Technology, 3359); anti-p-SIK3 (Abcam, ab225633); anti-SIK3 (Abcam, ab227044); anti-TYR (Santa Cruz Biotechnology, sc-20035); anti-GAPDH (Cell Signaling Technology, 5174); and anti-histone H1 (Santa Cruz Biotechnology, sc-8030). Secondary antibodies were horseradish peroxidase (HRP)-labeled anti-mouse IgG (Thermo Fisher Scientific, 31430); and HRP-labeled anti-rabbit IgG (Thermo Fisher Scientific, 31460).