Horseradish peroxidase conjugated goat anti mouse secondary antibody

Cells were lysed with ice-cold lysis buffer supplemented with protease inhibitor cocktail (Pierce; Thermo Fisher Scientific, Inc.). Total protein concentration was determined using the Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Protein samples (20 µg) were separated on a 10% SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane (Roche Diagnostics GmbH, Mannheim, Germany). Following blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated overnight at 4°C with rabbit anti-human ZIK1 monoclonal primary antibody (1:1,000; Abcam, Cambridge, UK; cat. no. 107918) and mouse anti-human β-actin monoclonal primary antibody (1:1,000; Abcam; cat. no. 8226) primary antibodies. Subsequently, the membranes were incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:3,000; Abcam; cat. no. ab6789) for 1 h at room temperature. The membranes were visualized with Super Signal West Pico Chemiluminescent Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). β-actin was used as a loading control. The Lab Works Image Acquisition and Analysis Software (version 7.0; UVP, LLC, Phoenix, AZ, USA) was used to quantify the band intensities.

Total proteins were extracted from cell lines and fresh tissues using radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and centrifuged at 14,000 × g for 10 min at 4°C to obtain crude protein extracts. The concentration of the protein extracts was measured by BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Protein samples (50 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk at room temperature for 1 h and then incubated with the following primary antibodies: STK33 mouse anti-human monoclonal antibody (dilution, 1:1,000; cat no. ab57693) and β-actin mouse anti-human monoclonal antibody (dilution, 1:1,000; cat no. ab8226; both Abcam, Cambridge, MA, USA) at 4°C for ~12 h. The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:1,000; cat no. ab97040; Abcam) at room temperature for 2 h. The bands were visualized using enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA) and quantified by densitometry analysis using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 20 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto polyvinylidene fluoride membranes (MilliporeSigma) and blocked with 5% non-fat milk in TBS-Tween-20 (TBST; 0.1% Tween-20) at room temperature for 1 h. The membranes were then incubated at 4°C overnight with the following primary antibodies: Anti-CCND3 (1:1,000; cat. no. ab28283; Abcam) and anti-GAPDH (1:1,000; cat. no. ab181602; Abcam). Following the primary antibody incubation, the membranes were rinsed five times with TBST and incubated with the secondary antibodies: Horseradish, peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. ab205718; Abcam) and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000; cat. no. ab205719; Abcam) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) reagent and densitometric analysis was performed using ImageJ version 1.50d software (National Institutes of Health).