Horseradish peroxidase hrp labeled secondary antibody

Tissue samples and cells were lysed with pre-cold RIPA lysis solution (Beijing Applygen Technologies) in a glass homogenizer, and centrifuged at 12,000 g for 10 min. Protein concentration was measured using a BCA kit (Thermo Fisher Scientific). Proteins (35 μg/sample) were separated using polyacrylamide gel electrophoresis with 10% sodium dodecyl sulfate and transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk prior to overnight incubation at 4 °C with a primary antibody against VDBP (1:5000 dilution, cat. no. ab81307, Abcam), c-Jun N-terminal kinase (JNK) (1:1000 dilution, cat. no. #9252, Cell Signaling Technology), or phosphorylated JNK (p-JNK) (1:2000 dilution, cat. no. #9255, Cell Signaling Technology). The membranes were then incubated with an appropriate horseradish peroxidase (HRP)-labeled secondary antibody (Zhongshan Jinqiao Biotechnology) for 1 h. Protein bands of interest were visualized using an ECL kit (Thermo Fisher Scientific).

Mouse zona pellucida polypeptide solution and the 330–342 amino acid sequence of mouse zona pellucida 3 (ZP3) (NSSSSQFQIHGPR), with an analytical purity of 91.5%, were obtained from Hangzhou Zhongpi Biochemical Co., Ltd. (P00105). Freund's complete adjuvant (F5881) and Freund's incomplete adjuvant (F5506) were obtained from American Sigma company. Rabbit anti-mouse TGF-β1, TGF-βRII, and Smad2/3 polyclonal antibodies were obtained from Wuhan Boster Bioengineering Co., Ltd. (BA0290, BA0526, BA1395). TRIzol reagent was obtained from Thermo Fisher Technology (China) Co., Ltd. [lot number 267309]. MonScri RTIII All-in-One Mix with sdDNase was obtained from Mona Biotechnology Co., Ltd. (lot number 130449). Universal SYBR qPCR Master Mix Universal Real-Time PCR Kit was obtained from China Biosharp Company (lot number 70090100). Horseradish peroxidase- (HRP-) labeled secondary antibody was obtained from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (ZDR-5118). Whole protein extraction kit was obtained from Nanjing KGI Biotechnology Development Co., Ltd. (KGP2100). BCA protein quantitative detection kit was obtained from Shanghai Shenggong Biological Engineering Co., Ltd. (C503031). LEICADMLB2 binocular microscope and LEICARM2255 automatic rotary microtome were obtained from German LEICA company.