Ecl plus detection kit

The cells were lysed at 4 °C in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts (30 μg) of protein were separated electrophoretically using SDS-polyacrylamide gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk, the membrane was incubated with specific antibodies (RAB3C: GTX108610, 1:5000, GeneTex, Taipei, Taiwan; RAB27A: GTX109180, 1:5000, GeneTex, Taipei, Taiwan; RAB3B: GTX108610, 1:5000; GeneTex, Taipei, Taiwan; RAB26: GTX118872, 1:5000; GeneTex, Taipei, Taiwan; IL-6: GTX110527, 1:1000, GeneTex, Taipei, Taiwan; STAT3: #4904, 1:1000, Cell Signaling, USA; phospho-STAT3: #9145, 1:1000, Cell Signaling, USA) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The blots were visualized using an ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).

Liver and testicular tissues at 25~50 mg were lysed in RIPA buffer, and the protein concentration was determined with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Anti-rat Abs for tubulin, AGER1,cluster of differentiation 36 (CD36), GLO-1, p38 mitogen-activated protein kinase (p38MAPK), and RAGE were used at a dilution of 1:1000 to detect immunoreactive signals, except for CML (1:100), tubulin (1:5000), and CTSD (1:2000). Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, San Jose, CA, USA), except for GLO-1 (Abcam, Cambridge, UK), RAGE (Abcam), CML (Abcam), tubulin (GeneTex, Hsinchu City, Taiwan), and CTSD (Sigma-Aldrich, Taipei, Taiwan). Blots were visualized using an enhanced chemiluminescence (ECL)-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).

Cells were lysed using RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 5 mM EDTA (pH 8.0), and 1 mM EGTA supplemented with protease and phosphatase inhibitors. After 20 min of lysis on ice, cell debris was removed via microcentrifugation and the supernatants were rapidly frozen. The protein concentration was measured via the Bradford method. In my experiments, equivalent samples containing 25–100 µg of protein were loaded onto an SDS-polyacrylamide gel, separated by electrophoresis, and electrophoretically transferred from the gel onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk, the membrane was hybridized with specific primary antibodies overnight at 4 °C and subsequently incubated with a corresponding horseradish peroxidase-conjugated secondary antibody for 1 h. The relative levels of proteins on the membranes were determined using an ECL-Plus Detection Kit (PerkinElmer Life Sciences, Boston, MA, USA).

The cells were lysed at 4 °C in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 5 mM EDTA (pH 8.0), and 1 mM EGTA supplemented with protease and phosphatase inhibitors. After 20 min of lysis on ice, the cell debris was removed via microcentrifugation, followed by rapid freezing of the supernatants. The protein concentration was determined using the Bradford method. In our experiments, equivalent loads of 25–50 μg of protein were electrophoresed using a SDS-polyacrylamide gel and then electrophoretically transferred from the gel to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated in specific primary antibodies (Ghrelin: GTX10473, GeneTex, Irvine city, CA, USA, 1:1000; Aurora A: #4718, Cell Signaling, Danvers, MA, USA, 1:1000; MMP10: sc-80197, Santa Cruz, Dallas, TX, USA, 1:1000; β-actin: A5316, Sigma-Aldrich, Louis, MO, USA, 1:5000; GHS-R 1: sc-374515, Santa Cruz, 1:2000) overnight at 4 °C and subsequently incubated in a corresponding horseradish peroxidase-conjugated secondary antibody for 1 h. The membranes were visualized using the ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).

Cells were lysed at 4 °C in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated by using sodium dodecyl sulfate polyacrylamide gels, and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated with specific antibodies overnight at 4 °C and then incubated with horseradish peroxidase (HRP) conjugated secondary antibody for 1 h. The blots were visualized by using an ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA) [31 (link)].

The cells were lysed at 4 °C in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of protein (30 μg) were electrophoretically separated using SDS-polyacrylamide gels and were then transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated overnight at 4 °C with the following specific antibodies: ALDOA: Cat. T0891, 1:1000 (Abcam (Epitomics), Cambridge, UK); ALDH1A3: Cat. GTX10784, 1:5000 (GeneTex, Taipei, Taiwan); Oct4: Cat. ab19857, 1:1000 (Abcam, Cambridge, UK); Nanog: Cat. 4903, 1:1000 (Cell Signaling, MA, USA); CD44: Cat. 3570, 1:1000 (Cell Signaling, MA, USA); BTK: Cat. sc-81159, 1:1000 (Santa Cruz, TX, USA); and Nestin: Cat. 66259-1, 1:1000 (Proteintech, IL, USA). Subsequently, the membranes were incubated with a horseradish peroxidase conjugated secondary antibody for 1 h. The blots were visualized using an ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).