Chemiluminescent imager

SHG-44 and U251 cells were lysed, and the protein quality was detected by BCA Protein Detection Kit (HyClone-Pierce, South Logan, USA). The 10-µg protein was separated by SDS-PAGE (Invitrogen, Carlsbad, CA, USA), transferred to the PVDF membrane, and sealed with TBST solution. Afterward, the proteins with the primary antibody (Table S1) were incubated at 37°C for 2 h, and then with the secondary antibody at 4°C overnight. Finally, Millipore Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Cat. No. RPN2232, Bedford, MA, USA) was used for color rendering and Chemiluminescent imager (GE, Cat. No. AI600, Chicago, IL, USA) observation.

The total proteins of MNNG/HOS and U-2OS were extracted with cell lysate, and detected by BCA protein detection kit (HyClone-Pierce). The 10-µg protein was separated by SDS-PAGE (Invitrogen) and transferred to the PVDF membrane, then sealed at room temperature for 1 h with TBST solution. After that, the membrane was first incubated with primary antibodies (Additional file 6: Table S1) overnight at 4 °C, and with HRP-conjugated goat anti-rabbit IgG for another 2 h at room temperature (The antibody information was shown in Additional file 6: Table S1). Finally, Millipore Immobilon Western Chemiluminescent HRP Substrote kit (Millipore, Cat. No. RPN2232) was used for color rendering and Chemiluminescent imager (GE, Cat. No. AI600) observation.