Hpa028885

Cells were lysed by RIPA buffer containing protease inhibitor cocktail (MedChem Express). Protein concentrations were determined by the BCA Protein Assay Kit (KeyGEN BioTECH). An equal amount of protein was loaded and separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories). Membranes were incubated with primary antibodies against PHF5A (1:400; Sigma, HPA028885), TEA domain family member 2 (1:500; Abcam, ab92279), HA-Tag (1:1,000; Cell Signaling Technology, 3724s), FLAG Tag (1:1,000; Sigma, F1804), and β-tubulin (1:1,000; Proteintech, 10068-1-AP) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h.

Paraffin-embedded sections were deparaffinized and rehydrated, followed by antigen retrieval. The sections were incubated with primary antibodies against PHF5A (1:50; Sigma, HPA028885) and Ki67 (1:200; Abcam, ab16667), followed by horseradish peroxidase-conjugated secondary antibody. The slides were finally incubated with diaminobenzidine (Dako) and counterstained with hematoxylin. For the 88 cases of CRC tissues, staining strength was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), and distribution was scored as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%) by positive staining area. The final score of PHF5A in each sample was obtained by multiplying the strength score by the distribution score. For the tissue microarray, the assessment of PHF5A staining was based on the percentage of positively stained cells and staining intensity using Image-scope software (Aperio Technologies).