Ab108600

Proteins were extracted in M-PER (Pierce) or RIPA buffer. Co-immunoprecipitation (Co-IP) experiments, with or without dithiobis(succinimidyl propionate) (DSP) (Thermo Scientific, Waltham, MA) were performed by incubating 1 mg of total proteins with HSP60 (Abcam, ab109660), HSP10 (Abcam, ab108600), Fhit^{48 (link)}, and HA antisera conjugated with protein A/G ultralink resin (Thermo Scientific) overnight at 4 °C; after TBST washing, beads were boiled in 1× reducing sample buffer and proteins were separated on polyacrylamide gel, transferred to a membrane, and probed with specific antisera.

Tissues and cells were lysed in IP lysis buffer (Beyotime, Jiangsu, China), and 500 μg samples of total protein were incubated with specific antibodies and protein A/G PLUS agarose beads (Santa Cruz, CA, USA)) overnight at 4 °C. The beads were washed with IP lysis buffer, and western blotting was then performed. The antibodies used were anti-Myc (9B11) mouse mAb (#2276S, Cell Signaling Technology, MA, USA), anti-HSPB1 (D6W5V) rabbit mAb (#95357, Cell Signaling Technology, MA, USA), anti-NPM1 rabbit pAb (10306-1-AP, Proteintech, IL, USA) and anti-HSPE1 rabbit mAb (ab108600, Abcam, MA, USA).

Immunohistochemical staining of 5-μm-thick consecutive sections of formalin fixed, paraffin-embedded tissue specimens was performed as previously described [79 (link)]. Antigens for HSPE1 were retrieved with Bond Epitope Retrieval Solution 1 using a Bond-Max automated immunostainer (Vision BioSystems, Melbourne, Australia) at 100°C for 20 min. Staining was performed with a specific antibody against HSPE1 (1:10000, ab108600; Abcam) at room temperature for 60 min using a standard protocol on the Bond-Max automated IHC stainer (Leica Biosystems, Global). A Bond Polymer Refine detection system (Vision BioSystems, Melbourne, Australia) was used to reduce nonspecific staining. Immunohistochemical staining was evaluated using a quantitative scoring method defined by two parameters: staining intensity (I) and percentage of positive-stained cells (P). For staining intensity, a score of 0 represents no staining, whereas scores of 1, 2 and 3 represent weak, moderate and strong staining, respectively. The final score was obtained by multiplying the intensity (I) by the percentage of positive-stained cell (P). HSPE1 protein expression levels in BC tissue and adjacent normal cells were categorized into three groups: low staining (score, 0–99), moderate staining (score, 100–199) and strong staining (score, ≥200).