High sensitivity ecl chemiluminescence detection kit

Total protein was released from cells through lytic effect by using ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China) with 0.5 mM/mL phenylmethylsulfonyl fluoride (Solarbio, Beijing, China). Lysate was centrifuged at 13,000 × g at 4°C for 15 min. The protein supernatant was transferred in a new tube and was incubated in 98°C with Protein Buffer (TransGen Biotech, Beijing, China) for 5 min. Same amount of protein was separated in a 12% SDS-PAGE, and transferred to PVDF membranes (BioRad, CA). QuickBlock Blocking Buffer (Byotime, Shanghai, China) was used to membranes incubation for 30 min at room temperature. Blocked membranes were incubated with antibodies against MyoD (1:500, NovusBio, CO), MyHC (1:200, DHSB, IA), MurF-1 (1:500, Proteintech, IL), Atrogin-1 (1:1000, Abcam, Cambridge, UK) and GAPDH (1:2000, Bioworld, MN) overnight at 4°C. Next, anti-mouse or anti-rabbit IgG secondary antibodies with HRP label were used to incubate the membranes for 1 h at room temperature. High sensitivity ECL chemiluminescence detection kit (Vazyme, Nanjing, China) was used in chromogenic reaction by following the instruction. Chromogenic bands were captured in an Odyssey instrument (Li-cor, CA). The gray value of bands was measured in ImageJ software.

Total proteins were extracted from SK-N-SH and SK-N-BE cells using RIPA lysis buffer (Beyotime, Shanghai, China). Equal amount (20 µg) of total proteins were separated by gel electrophoresis with 12% SDS-polyacrylamide gel (SDS-PAGE), and then transfected onto the PVDF membranes (polyvinylidene fluoride; Millipore, Bedford, MA, USA). Subsequently, membranes were incubated with Tris-Buffered Saline Tween-20 (TBST; Solarbio, Beijing, China) supplemented with 5% bovine serum albumin for 1 h at room temperature. Then the membrane was cultivated overnight at 4 °C with primary antibodies purchased from Abcam (Abcam, Cambridge, MA, USA) which was diluted in TBST solution. The primary antibodies used in this research were shown as below: Cleaved-caspase 3 (Cleaved-casp-3; 1:500, ab13847), B-cell lymphoma-2 (Bcl-2; 1:1000, ab32124), Bcl-2-Associated X (Bax; 1:7000, ab32503), GPNMB (1:100, ab227695), PCNA (1: 1000, ab18197) and GAPDH (1:1000, ab8245). Subsequently, the membranes was washed with TBST for three times and then incubated with corresponding secondary antibody Goat Anti-Rabbit IgG H&L (1: 20,000, ab205718, Abcam) for 1 h at room temperature. Then the bands were visualized using High sensitivity ECL chemiluminescence detection kit (Vazyme, Nanjing, China) and quantified by Quantity One software version 4.6 (Bio-Rad Laboratories). GAPDH was used as the internal control.

Lysis buffer (Beyotime) supplemented with 1% phenylmethylsulfonyl fluoride (Beyotime) was used to extract total protein from cells. Cell extracts were centrifuged at 12,000g for 15 min, and the supernatant was collected. Cell lysates were resolved on 8% acrylamide gradient SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk in tris-buffered saline containing 0.1% Tween 20 for 1 hour, then incubated with anti-BRD4 (1:1000 dilution; Abcam) and anti–β-actin (1:10000 dilution; Sungenebiotech) antibody followed by horseradish peroxidase–conjugated secondary antibody, and detected by immunoblotting with the High-Sensitivity ECL Chemiluminescence Detection Kit (Vazyme) or Super ECL Detection Reagent (Yeasen Biotech).