Ecl western detection kit

Immunoblot analysis was performed as described previously [47 (link)]. For protein extraction, leaf samples were ground into powder in liquid nitrogen and homogenized in extraction buffer (100 mM HEPES, pH 7.5; 5 mM EDTA; 5 mM EGTA; 10 mM Na₃VO₄; 10 mM NaF; 50 mM β-glycerophosphate; 1 mM phenylmethylsulphonyl fluoride; 10% (v/v) glycerol; 7.5% (w/v) polyvinylpolypyrrolidone; and 0.2% (v/v) β-mercaptoethanol), followed by centrifugation at 12,000× g for 20 min. Proteins adjusted to equal concentration were then mixed with 4× loading buffer and denaturized at 95 °C for 10 min. SDS–polyacrylamide gel (12% [w/v]) electrophoresis (SDS-PAGE) was performed to separate the protein extracts. For HY5 detection, we used an HY5-specific antibody (Shanghai Jiayuan Bio Co., Shanghai, China), which was detected using the ECL western detection kit (GE Healthcare, Boston, MA, USA). HSP70 antibody (Beijing Protein Innovation Co., Ltd., Beijing, China) was used as a control.

The vector pER8-CgNLP1-FLAG was constructed with an estradiol-inducible promoter and then transformed into Agrobacterium tumefaciens GV3101. The Agrobacterium-mediated flower dip method was used for Arabidopsis transformation of CgNLP1. T1 transgenic lines were screened with 30 mg/L hygromycin, and Western blot was used to confirm the positive transgenic lines after estradiol treatment. Appropriate total proteins from different lines were resolved on 10% polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad). Anti-FLAG monoclonal antibody (1:1,000, ab125243; Abcam) was used as a primary antibody to detect the expression of CgNLP1 in transgenic lines. Horseradish peroxidase-conjugated anti-mouse antibody was used as the secondary antibody, and the signal was detected using the ECL western detection kit (RPN2232; GE Healthcare).

Cytosolic proteins were isolated from an aliquot of frozen pulverized xenograft tumors used for miRNA- 126 real-time qRT-PCR analysis. Western blot analysis was done as described 63 (link) using equal amounts of cytosolic protein extract (30 µg) isolated from MDA-MB-231 and Panc1 CSC-derived xenograft tumors from test and control tumors. Cytosolic extracts were prepared by tissue homogenization in buffer containing 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 250 mM sucrose, protease inhibitors, followed by centrifugation (11000g x 10 min) and final collection of supernatant (cytosolic fraction). Proteins were separated on a 15% SDS-PAGE and were transferred to PVDF membrane (Bio-Rad). The Western blot was reacted sequentially with anti-KRAS (Abcam cat# ab55391 at 20 µg/mL) followed by anti-b-actin (Santa Cruz cat# sc-47778 at 1 µg/mL) antibodies. Immunoreactive proteins were detected by chemiluminescence using the ECL Western Detection kit (GE Healthcare).