Ecl plus western blotting detection kit

Proteins for each sample (20 μg) were resolved on 4–20% pre-cast gradient SDS-PAGE gels (Life Technologies, NY), and then electro-transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies overnight in 4°C, followed by horseradish peroxidase-labeled second antibody (Santa Cruz Biotech, CA) for 1 h at room temperature. ImageJ software (NIH) was used to measure band intensities. Primary antibodies were—Anti-Actin (Santa Cruz Biotech, CA); Anti-SRB1 (Pierce Biotech, IL), anti-Akt, anti-Phosho-Akt, anti-PI3K, and anti- ABCA1 (Millipore, CA). Finally, detection procedures were performed using an ECL Plus Western Blotting Detection Kit (Amersham, Piscataway, NJ).

Jurkat, HT-29 and HEK293 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Jurkat cells were cultured as described in Ref 4 (link) while HT-29 and HEK293 cells as reported in Ref 44 (link). Antibodies against p21^waf1/cip1, β-actin and caspase 3 precursor were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); AKT, p-AKT and PARP from Cell Signaling (Danvers, MA, USA), anti-PPARγ (ab59256) and anti-Hsp60 (ab46798) from Abcam (Cambridge, United Kingdom); anti-mouse and anti-rabbit IgG peroxidase-linked secondary antibodies, ECL and ECL Plus Western blotting detection kit from Amersham Life Science (Little Chalfont, Buckinghamshire, UK). Dulbecco’s Modified Eagle’s Medium (D-MEM), FBS, penicillin-streptomycin, l-glutamine, trypsin-EDTA and OptiMEM I were from Gibco (Carlsbad, CA, USA), charcoal/dextran-treated FBS was from Hyclone (Logan, Utah, USA).

Protein concentrations of mouse testes lysates were measured using the micro-BCA method (Pierce, Milwaukee, WI). The extracted proteins (25 μg) were loaded on 12% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond-P, Helsinki, Finland). The nonspecific binding was blocked with PBS buffer containing 0.1% Tween-20, 2% BSA, and 5% nonfat dry milk, and the blots were then incubated with anti-rabbit CHD1L (Abcam), anti-goat GFRΑ1 (Santa Cruz, Dallas, Texas), anti-goat PLZF (Santa Cruz), or anti-rabbit β-actin (Santa Cruz), respectively. After extensive washing with 0.1% Tween-20 in PBS, the blots were incubated with horseradish peroxidase-conjugated anti-goat (Santa Cruz) or anti-rabbit (Santa Cruz) IgG at room temperature for 1 h. Visualization was developed using ECL Plus Western Blotting Detection Kit (Amersham, Helsinki, Finland). Quantification of blot intensities was performed using Image Lab software (Bio-Rad, Hercules, California), according to the developer's protocol. β-Actin was immunoblotted and visualized as a loading control. The antibodies used and their concentrations are listed in Supplemental Table 2.

Liver tissues (100 mg) were lysed in 100 mL of RIPA lysis buffer in the presence of PMSF (10 mL). 25 μg of protein was separated on a SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was blocked with 3% non-fat milk in PBS-T and incubated with rabbit anti-LC3 antibody (1:2000, abcam, USA), anti-Omi antibody (1:2000, abcam, USA), LAMP2 antibody (1:2000, abcam, USA) and Beclin-1 antibody (1:2000, abcam, USA) overnight at 4°C, respectively. The secondary antibody used was sheep anti-rabbit IgG-HRP antibody. The bands were detected using the ECL Plus Western blotting detection kit (Amersham, USA).