The cells were lysed on ice with the RIPA (Radio Immunoprecipitation Assay) lysis buffer (Thermo Fisher Scientific, USA) for 30 min to prepare the cell suspension, followed by centrifugation at 14,000 rpm for 10 min at 4 °C. The protein concentration was determined with bicinchoninic acid protein assay (Thermo Fisher Scientific, USA), 0.1 mg total protein were subjected to 10% SDS-PAGE separation and then transblotted to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked in 5% nonfat milk for 1 h at room temperature, and then incubated with the primary antibodies. Target proteins were detected by using specific antibody against CALM1 (catalog number: #10,541–1-AP; dilution at 1:800; Proteintech Group, Wuhan, China). GAPDH (catalog number: 10494–1-AP; dilution at1:5000, Proteintech Group, Wuhan, China) was chosen as an internal control and the CALM1 and GAPDH dilutions were incubated at 4 °C with gentle shaking overnight. Then, secondary antibody (goat anti-rabbit, catalog number:SA00002-2, Proteintech Group, Wuhan, China) were added onto the membrane for incubation at room temperature for 2 h.The blots were visualized with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, CA, USA), according to the manufacturer's protocol.

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