Lysis buffer

The testes or cells were lysed with a lysis buffer (Applygen Technologies Inc., Beijing, China), and the protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The proteins (20 μg/well) per well were separated on 10% SDS-PAGE gel and then electrotransferred onto PVD membranes (Millipore, Bedford, MA, USA). The membranes were blocked with Tris-buffered saline (TBS, pH 7.4) containing 5% non-fat milk at room temperature for 1 h and then incubated with the primary antibodies overnight at 4 °C. The membranes were washed twice with TBS containing 0.1% Tween-20 and then incubated with the horseradish-peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Ag/Ab complexes were visualized using an enhanced chemiluminescence detection kit (Zhongshan Biotechnology Co.). β-Actin was used as the loading control. Band densitometry was quantified using ImageJ software (http://rsb.info.nih.gov/ij).

Cells were harvested and lysed in nondenaturing lysis buffer (Applygen Technologies, Beijing, China). Equal quantities (40 µg of protein) of cell extract were resolved by 10% SDS–PAGE, and the resolved proteins were electrophoretically transferred to PVDF membrane and blocked with 5% fat-free dry milk in TBST for 2 h at room temperature. Then respective primary antibodies were added in 5% milk TBST and incubated overnight at 4°C and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature and washed three times before detection. The blot was developed with LAS4000 enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK) and the densities of the bands were determined using Gel-Pro Analyzer 4.0 software.

Cells and tissues were first lysed using lysis buffer (Applygen, Beijing, China) with a phosphatase and protease inhibitor cocktail (Roche, Branchburg, NJ, USA). Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate the different proteins. Polyvinylidene difluoride (PVDF) membranes were blocked with 5% nonfat milk for 3 h and subsequently incubated with antibodies at a temperature of 4 °C for a duration of 10 h. Subsequently, the PVDF membranes were incubated with the appropriate IgG-HRP conjugate and the resulting protein bands were analyzed using a Bio-Rad ChemiDocTM XRS+ imaging system (Bio-Rad Laboratories). All antibodies used for this research are listed in Table S4.

Total protein was extracted from NFPAs or normal pituitaries using lysis buffer (Applygen, Beijing, China) following the manufacturer’s instructions. The protein concentration was measured using the bicinchoninic acid method. Protein samples (30 μg) were separated by electrophoresis using 10% sodium dodecyl sulfate polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20, and incubated with rabbit antibodies against human Smad2 (1:1000; Cell Signaling Technology, Boston, MA, USA), p-Smad2 (Ser465/467) (1:1000; Cell Signaling Technology, Boston, MA, USA), Smad3 (1:1000; Cell Signaling Technology, Boston, MA, USA), p-Smad3 (Ser423/425) (1:1000; Cell Signaling Technology, Boston, MA, USA), and GAPDH (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the membranes were incubated with the secondary antibody anti-rabbit IgG (1:4000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the membranes were developed using an ultrasensitive chemiluminescence protein dye detection system (ECL Plus; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and exposed to X-ray films (Kodak, Rochester, NY, USA). The bands were subjected to grayscale scanning and semi-quantitative analysis using Quantity One software (Bio-Rad, Hercules, CA, USA).

Frozen muscle tissue or spinal cord (L6-S1) tissue was homogenized in ice-cold lysis buffer (Applygen Technologies Inc., Beijing, China) with protease inhibitor cocktail (Roche, Switzerland). Equal amounts (50 μg) of extract were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, United States) at 4°C. Non-specific binding sites were blocked with 10% non-fat milk in TBS (20 mM Tris-Cl, pH 7.5, containing 0.15 M NaCl and 2.7 mM KCl) for 1 h at room temperature. The membrane was incubated with the primary antibodies listed in Table 1 overnight at 4°C. After washes with TBST, the membrane was incubated with the appropriate secondary antibodies goat anti-rabbit IgG (1:10,000, 611-132-122, Rockland, United States) or sheep anti-mouse IgG (1:10,000, 610-632-002, Rockland, United States) for 2 h at room temperature. The membrane was washed in TBST and visualized with an Odyssey Infrared Imager (LI-COR, NE, United States). The integrated intensity of the bands was analyzed with Odyssey software (version 1.2).

Dissected livers of zebrafish larvae (20–30 in each group) were washed with ice-cold PBS and lysed directly by lysis buffer (Applygen Technologies Inc., Beijing, China). The protein samples were blotted and incubated with ATG3 (SC-70139, Santa Cruz Biotechnology, Texas, USA), LC3B (PM036, MBL, Japan), p62 (PM045, MBL, Japan), β-actin (A5441, Sigma-Aldrich) and secondary HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (ZSGB-BIO, Beijing, China). The protein bands were detected with SuperSignal West Pico chemiluminescent substrate (34080, Thermo Fisher, Waltham, MA).