Ab131021

After treatments, NPC cells were washed with PBS and then lysed with cell lysis buffer (CST, USA). Whole cell lysates were centrifuged at 14,000g for 5 min at 4 °C to remove cell debris, and the supernatants were subjected to SDS-PAGE and immunoblotting, as previously described [25 ]. The following antibodies used in this study were purchased from Abcam (USA): rabbit anti-TET1 (ab191698), rabbit anti-TET2 (ab94580), rabbit anti-TET3 (ab139311), mouse anti-GAPDH (ab8245), rabbit anti-flag (ab1162), rabbit anti-PKM antibody (ab131021), and rabbit anti-Histone H3 antibody (ab176842).

IHC was performed on 5-μm-thick sections from formalin-fixed, paraffin-embedded tissue samples applied to coated slides. The procedures of IHC staining were performed as we previously described [18 (link), 19 (link)]. The following antibodies including HSP90 (Abcam, ab13492) and PKM2 (Abcam, ab131021) were used for IHC staining of HCC tissues.

Co-immunoprecipitation was carried out using the Thermo Scientific Pierce Co-IP Kit (26149) following the manufacturer’s protocol. Briefly, anti-VDAC2 (ab126120, Abcam), anti-PFKP (ab204131, Abcam), or an IgG control antibody (mAb#3900, Cell Signaling Technology) was immobilized for 2 h using AminoLink Plus coupling resin. The resin was then washed and incubated with cell lysate overnight. After incubation, the resin was washed again and proteins were eluted using elution buffer. Samples were analyzed by western blot using antibodies, including anti-VDAC2 (ab126120, Abcam), anti-PFKP (ab204131, Abcam), anti-PKM2 (ab131021, Abcam), anti-LDHA (#3582, Cell Signaling Technology), and anti-GAPDH (ab181602, Abcam).