Pa550622

After treatment, cells were lysed with RIPA lysis buffer (Abcam, Cambridge, UK), and total protein was purified by a standard protocol. Protein concentration was determined by the BCA Protein Assay Kit (Abcam, Cambridge, UK). Before SDS-PAGE separation, the samples were denatured by boiling for 5 min and then kept on ice. The separated proteins were transferred from the gel to a PVDF membrane by a semidry system. The membrane was blocked by incubation in 3% BSA solution, incubated with primary antibodies, washed, and incubated with IRDye® infrared dye-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany). The infrared intensity was measured with an Odyssey CLx Infrared Imaging System (LI-COR). Rabbit polyclonal antibodies against UHMK1 (PA550622, Invitrogen, Germany), GAPDH (Cell Signaling Technology, Danvers, MA, USA), and IRDye^® 800CW goat anti-rabbit IgG secondary antibody (LI-COR) were used.

Immunohistochemistry on 6-µm frozen or paraffin-embedded tissue sections was performed as previously described (41 (link)). Primary antibodies included rabbit polyclonal antibodies against UHMK1 (PA550622, Invitrogen, Germany) and Ki67 (Abcam, Cambridge, UK). Goat anti-rabbit biotinylated IgG (Vector, Burlingame, CA, USA) was the secondary antibody. ImageJ was used to calculate the intensity of the signal emitted from positively stained cells.