Anti muc1 antibody

The expression of MUC1 was assessed by flow cytometry. The cells were fixed and permeabilized using cytofix/cytoperm^TM (BD Biosciences, San Jose, CA, USA). After washing, the cytofix/cytoperm^TM wash solution (BD Biosciences) was added to the cells together with anti-MUC1 antibody (Abcam, Cambridge, MA, USA) recognizing the C-terminal domain of MUC1 and incubated at 4℃ overnight. The cells were washed with the cytofix/cytoperm^TM wash solution and then incubated with the Alexa 488-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) in cytofix/cytoperm^TM wash solution at 4℃ in the dark for 1 hr. Samples were analyzed by a FACSCalibur (BD Biosciences) flow cytometer and the data were analyzed with the software FCS Express 5 (De Novo Software, Pasadena, CA, USA).

The supernatant after cell lysis was collected with radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The concentration of proteins was measured by the bicinchoninic acid Protein Assay Kit (Sangon Biotech), and the same amount of protein was denatured with 5 × sodium dodecyl sulfate (SDS; Sangon Biotech) loading buffer in boiling water at 100°C for 5 min. To separate the proteins, SDS-polyacrylamide gel electrophoresis was used; then the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and the PVDF membrane was incubated with 5% non-fat milk at room temperature for 1 h. Next, the anti-MUC1 antibody (1:1000; Abcam) and GAPDH (1:1000; Abcam) were added and incubated at 4°C overnight. Then the membrane was washed with Tris-buffered saline with Tween-20 three times; subsequently, the secondary antibody (HRP-labeled) was added and incubated at 20°C for 1 h. Finally, the protein was tested using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).