Anti hrd1

MCF7 and MCF7/Tam cells were washed with ice-cold PBS for 2 times, and then were added with RIPA lysis buffer. Protein in each sample was analyzed by western blotting as previously reported [7 (link)]. The dilution of each antibodies is: anti-S100A8 (1:500) (Proteintech, Chicago, USA), anti-HRD1 (1:1000) (Abcam, Cambridge, UK), anti-GAPDH (1:4000) (Abcam, Cambridge, UK), anti-HA-tag (1:1000) (Proteintech, Chicago, USA), anti-His-tag (1:1000) (Santz Cruz, CA, USA), anti-Myc-tag (1:1000) (Proteintech, Chicago, USA).

Fifty percent confluent cells were cultured on glass coverslips in the wells of 24-well plates for 24h. Then cells were washed, fixed, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with goat serum for 2 h, cells were incubated for 1 h with antibody anti-S100A8 (1:30dillution) (Proteintech, Chicago, USA) and Anti-HRD1 (1:50 dilution) (Abcam, Cambridge, UK). Then, dishes were washed and incubated with Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibodies (1:50 dilution) for 1h at room temperature. Nuclei were stained with Hoechst (10 mg/ml) for 2 min. Samples were examined with fluorescence microscope (Zeiss, Oberkochen, German).