Cells, cell pellet and EVs pellet were lysed with RIPA (Radio Immunoprecipitation Assay) lysis buffer (P0013B, Beyotime, Shanghai, China) containing 1 mM PMSF (Phenylmethanesulfonyl fluoride) (ST506, Beyotime, Shanghai, China). Proteins were separated by 8%–20% acrylamide/bisacrylamide gels and transferred to PVDF membranes (Millipore, Billerica, USA). The proteins were blocked in 5% blocking regent (1×TBST containing 5% defatted milk powder) for 1 h at room temperature. Membranes were incubated with primary antibodies (1:1000–1:3000) in antibody dilution buffer (G2025, Servicebio, Wuhan, China) for overnight at 4°C. After that, membranes were washed with 1×TBST for three time and then incubated with HRP (horseradish peroxidase)-conjugated second antibodies (1:1000–1:5000) in antibody dilution buffer for 1 h at room temperature. The proteins were visualized with Tanon 6200 (Tanon, Shanghai, China). The following antibodies were used, rabbit anti-human VPS28 (Proteintech, 15478-1-AP), rabbit anti-human Calnexin (Proteintech, 10427-2-AP), rabbit anti-human TSG101 (Proteintech, 28283-1-AP), rabbit anti-human CD63 (Proteintech, 25682-1-AP), anti-human β-Actin (Proteintech, 66009-1-Ig) and anti-mouse VEGF (Santa cruz, sc-7269).
G2025
Manufactured by Wuhan Servicebio Technology
The G2025 is a laboratory equipment produced by Wuhan Servicebio Technology. It is designed for cell culture and related applications. The core function of the G2025 is to provide a controlled environment for cell growth and maintenance.
Automatically generated - may contain errors
2 protocols using g2025
1
Western Blot Analysis of Cellular and EV Proteins
2
Protein Expression Analysis in Cell Lysates
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Cells were lysed in RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA-Na2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors and phosphatase inhibitor on ice for 15 min. The cell lysates were cleared by centrifugation at 12,000×g for 10 min, then heat-denatured with 5 × Laemmli buffer (G2013, Servicebio). The prepared samples with equal amount of total protein (20 μg) were subjected to SDS-PAGE and immunoblotting, as per the standard procedure. The following antibodies were used: anti-SOX1 (ab109290, Abcam), anti-β-catenin (ab32572, Abcam), anti-HES1 (#11988, Cell Signaling Technology [CST]), anti-PROX1 (11067-2-AP, Proteintech), anti-p38 (#8690, CST), anti-phospho-p38 (#9215, CST), anti-ERK (#4695, CST), anti-phospho-ERK (#4370, CST), anti-AKT (#4691, CST), anti-phospho-AKT (#4060, CST), anti-JNK (#9252, CST), anti-phospho-JNK (#700031, Invitrogen), anti-RAF (#9422, CST), anti-phospho-RAF (#9427, CST), anti-MEK (51080-1-AP, Proteintech), anti-phospho-MEK (#9154, CST), anti-α-Tubulin (11224-1-AP, Proteintech), anti-GAPDH (BM1623, Boster), anti-BCL2 (Proteintech, 12789-1-AP), anti-PCNA (BM0104, Boster). All antibodies were diluted by antibody dilutions (G2025, Servicebio) with a ratio of 1:1,000. All quantitative analyses were performed using the software image J.
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