Rabbit anti beclin1

ApoG2, obtained from Xi’an Jiaotong University (Xi’an, China), was prepared at a stock concentration of 0.20 mmol/l in dimethyl sulfoxide (DMSO). RPMI-1640 medium, L-glutamine, trypsin-ethylenediaminetetraacetic acid, penicillin/streptomycin and fetal bovine serum (FBS) were obtained from HyClone Laboratories (Logan, UT, USA). The reagents used for the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were purchased from Boehringer Mannheim (Mannheim, Germany). Acridine orange was purchased from Molecular Probes (Eugene, OR, USA). Rabbit anti-LC-3B and rabbit anti-beclin-1 were purchased from Abcam (Cambridge, UK). MTT, 3-methyladenine (3-MA), annexin V-fluorescein isothiocyanate (FITC), propidium iodide (PI), Hoechst 33342, formaldehyde, HEPES, sodium pyruvate, glucose and DMSO were obtained from Sigma (St. Louis, MO, USA). All other reagents are commercially available and were of analytical grade.

The neurons grown on coverslips were fixed by 4% methanol for 15 min and 0.25% Triton for 10 min, incubated in 2% Bovine Serum Albumin(Sigma) for 1 h to block nonspecific binding of IgG. Then the cells were reacted with primary antibodies: rabbit anti-Beclin1 (1:200, Abcam), rabbit anti-BCL-2 (1:200, Santa Cruz), mouse anti-NeuN (1:200, Millipore) at 4°C overnight followed by the appropriate secondary antibodies with a mixture of goat anti-rabbit Alexa-Fluor 488 and goat anti-mouse Alexa-Fluor 594(1:200, Invitrogen). The cells were subsequently incubated with the nuclear staining dye 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, Burlingame, CA, USA). Finally, the stained cells were observed with a fluorescent microscopy (Nikon ECLIPSE 80i, Japan).

Kidney tissues (40 µg) were homogenized in 1 mL of RIPA Buffer (Solarbio, R0020, Beijing, China) containing 10 µL of phenyl methyl sulfonyl fluoride as a protease inhibitor. Next, proteins (60 µg) were separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Membranes were blocked with casein and subsequently incubated with the following primary antibodies at 4 °C overnight: anti-Wilms Tumor (anti-WT1, 1:1000, Abcam, USA), anti-Nephrosis2 (anti-NPHS2, 1:1000, Abcam, USA), rabbit anti-Beclin1 (1:1000, Abcam, USA), rabbit anti-LC3B (1:1000, Abcam, USA). Next, membranes were probed with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG for 1 h at room temperature (Beyotime, Beijing, China). Following washing, membranes were visualized using High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) and the protein bands were analyzed using densitometry with Image J.

To determine the expression levels of LC3B, and phosphor-Akt-ser473 (p-Akt-ser473), the samples were suspended in radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethylsulfonyl fluoride and phosphatase inhibitor (100:1:1) for protein extraction. The samples were then centrifuged at 12,000 rpm at 4°C for 30 min, and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the bicinchoninic acid protein assay kit (Sigma, MO, USA). Protein (40 μg) was electrophoresed on a precast bis-Tris polyacrylamide gel (12% or 10%), and transferred onto a polyvinylidene difluoride membrane. Membranes were blotted with rabbit anti-APN (1:1000), rabbit anti-LC3B (1:500), rabbit anti-P62, rabbit anti-beclin-1 (both from Abcam, USA), rabbit anti-p-Akt, rabbit anti-Akt (CST, USA), and mouse anti-actin (1:5000; BL005A; Biosharp, Beijing, China), followed by horseradish peroxidase-conjugated secondary antibodies (1:5000; ZsBio, Beijing, China). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000).

For immunoprecipitation (IP), the cells grown in 10 cm cell culture dishes were harvested and incubated in the precooled IP lysis buffer for 30 min at 4°C. The resulting mixture was centrifuged at 14000g for 15 minutes. The supernatant was collected and the protein concentration of the samples was estimated using the Bradford method. Equal amounts of protein samples were incubated with rabbit anti-Beclin-1 (Abcam, Cambridge, MA, USA) primary antibody in a 1:100 dilution at 4°C and mixed constantly by inversion for 2 h. Then, 5 μl of protein A/G magnetic beads (Bimake, Houston, TX, USA) were added to the lysate and incubated overnight in an inverted position at 4°C in a magnetic rack (Bimake, Houston, TX, USA). Then, the lysate with the magnetic beads were centrifuged. The supernatant was removed. Then, 40-60 μl of the loading sample buffer solution was added to the magnetic beads and boiled for 10 min. The liquid supernatant was stored at−80°C for electrophoresis.

Fresh bone metastasis tissues were harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology, Inc., CHN). Protein concentrations were measured using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Inc., CHN). The total protein of each specimen (30 mg/lane) was separated by SDS-PAGE (10% gels), and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Inc., USA). B-actin was used as a loading control. The membrane was blocked with 5% bovine serum albumin (BSA, Beijing Solarbio Science & Technology Co., Ltd., CHN) at room temperature for 40 min, and subsequently incubated with mouse anti-p62 (1:1,500; cat. no. ab56416; Abcam), rabbit anti- LC3B (1:1,000; cat. no. ab48394; Abcam, Inc., UK), rabbit anti-Beclin1 (1:1,000; cat. no. ab207612; Abcam, Inc., UK) and mouse anti-β-actin (1:5,000; cat. no. ab6276; Abcam, Inc., UK) primary antibodies overnight at 4°C. The membranes were then incubated with peroxidase-conjugated goat anti-mouse IgG (1:20,000; cat. no. A4416; Sigma-Aldrich; Merck KGaA, Inc., GER) at room temperature for 1 h. Protein bands were visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate reagents (Thermo Fisher Scientific, Inc., USA). The relative gray value of the immune reactive bands was compared using ImageJ software (version 1.46, National Institutes of Health, USA).