Nek6 antibody

Cells and tissue homogenates were harvested and lysed in RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM Na₃VO₄, and 1 mM NaF). The protein concentration in the lysate was quantified using the BCA Kit. The cell lysates were loaded onto an SDS-PAGE gel for electrophoresis. After transfer of the proteins onto a piece of nitrocellulose membrane, targets were detected by western blotting using primary antibodies, including NEK6 antibody (1 : 2000, catalog number: 10378-1-AP, Proteintech, Wuhan, China), GAPDH antibody (1 : 10,000, catalog number: 10494-1-AP, Proteintech), secondary antibody, HRP-conjugated goat anti-mouse IgG (H + L) (1:10,000, catalog number: ab205719, Abcam, United States), HRP-conjugated goat anti-rabbit IgG (H + L) (1:10,000, catalog number: ab205718, Abcam), and an ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, catalog number: WP20005, Shanghai, China). Protein expression levels were quantified using ImageJ software. The experiments were performed in triplicate.

Proteins in cells were extracted with precooled radioimmunoprecipitation assay buffer (catalog number: P0013C; Beyotime, Shanghai, China), and protein quantification was performed using a BCA protein concentration determination kit (catalog number: P0012S; Beyotime). Electrophoresis was performed on a 10% sodium dodecyl sulfate-polyacrylamide gel, and the proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (catalog number: IPFL00010; Millipore, Shanghai, China). After incubation with 5% skim milk at 25°C for 2 h, the PVDF membranes containing the proteins were incubated with diluted NEK6 antibody (catalog number: 10378-1-AP; dilution rate: 1 : 1000; Proteintech, Wuhan, China) or GAPDH antibody (catalog number: 10494-1-AP; dilution rate: 1 : 5000; Proteintech) at 25°C for 2 h. After washing, the PVDF membranes were incubated with diluted goat anti-rabbit IgG modified with horseradish peroxidase (catalog number: SA00001-2; dilution rate: 1 : 2000; Proteintech) at 25°C for 1 h. After washing, the PVDF membranes were covered with BeyoECL Moon (catalog number: P0018FM; Beyotime) for exposure. Quantification by densitometry was performed using ImageJ 1.52v (NIH, Bethesda, MD, USA). GAPDH was used as an internal control.