Hsa_circ_0136682 driving malignancy in breast cancer through the NCL/c-Myc axis

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the original picture of western blot

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Western blot Cells were collected from a 6-well plate and centrifuged. Protein lysates were harvested using RIPA buffer (Beyotime, Shanghai, China) and added PMSF (1%; Beyotime, Shanghai, China), then the protein concentration was assessed using a bicinchoninic acid (BCA) Kit (Yeasen, Shanghai, China). Equal amounts of protein were separated by SDS‒PAGE(10%), and transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5% nonfat milk, membranes were incubated with primary antibody at 4 °C overnight, washed with Tris-buffered saline-Tween 20 (TBST), and then incubated with secondary antibody for 45 min. The primary antibodies were NCL (1:1000; Proteintech, Wuhan, China), c-Myc (1:1000; Proteintech, Wuhan, China), Histone H3 (1:5000; Huaan, Hangzhou, China), Tubulin α (1:5000; Huaan, Hangzhou, China), and GAPDH (1: 5000; Proteintech, Wuhan, China). The membranes were reacted with ECL Substrate (Fdbio, Nanjing, China) and images were collected using Tanon 5200 (Nanjing, China).

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