Sc 100443

Expression of SRPK1 was assessed by a standard IHC method [28 (link)]. Anti‐human SRPK1 monoclonal antibody (1:500, sc-100443, Santa Cruz BioTechnology) was selected as the primary antibody, and anti‐mouse antibody (1:5000, ab6708, Abcam) was used as the secondary antibody. IHC data were evaluated by two independent pathologists who were blinded to patients’ information, a consensus was required to be obtained in case of divergence. The staining was assessed by both staining intensity and proportion of stained cells.
Accordingly, staining intensity was scored as no staining (0 point), weak (1 point), moderate (2 points), or strong (3 points). Proportion of the stained cells was classified as negative (0 point), <33% (1 point), 33%‐66% (2 points), or >66% (3 points). The final IHC staining score was obtained by the product of the two scores. An overall score of 3 or less was defined as low SRPK1 expression, and that of 4 scores or more was defined as high SRPK1expression.

Total protein was extracted from transfected cells using radioimmunoprecipitation assay buffer (Beyotime, China) supplemented with protease inhibitor cocktail (Roche, USA). After protein quantification with a bicinchoninic acid kit (Thermo Fisher Scientific, USA), equal amount of protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane using the Bio-Rad system (Bio-Rad, USA). After an overnight incubation with an anti-SRPK1 antibody (1:2000, sc-100443, Santa Cruz) or an anti-beta-actin antibody (1:2000, sc-47778, Santa Cruz) at 4°C, membranes were incubated with secondary antibody (1:5000, ab6708, Abcam) at room temperature for an additional 1 h. The protein expression level was finally evaluated by using the Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific) [29 (link)] using Tanon 4200SF Multi-Function Imaging System (Shanghai Tianneng Technology Co., Ltd., China). Finally, images were semi-quantified using the Image J Software (NIH, USA).