Chemiluminescent substrate reagent kit

Fluorescent proteins were visualized directly in the agarose gels using a UV transilluminator (Cleaver Scientific, Rugby, UK). Blotted membranes were stained with Ponceau S solution, and proteins were also detected by means of anti-HisTag-HRP (horseradish peroxidase) antibodies (Thermo Fisher, Cat No. MA1-80218, Waltham, MA, USA) in combination with Novex™ ECL (Invitrogen Chemiluminescent Substrate Reagent Kit, Cat No. WP20005, Waltham, MA, USA) as a reaction substrate. Membranes were imaged with a UVITEC chemiluminescence imaging system (Cambridge, UK). In situ APEX activity in agarose gels was detected in the presence of TMB (3,3′,5,5′-Tetramethylbenzidine) (VWR—Cat. No. J644-100 ML) until colored bands appeared. APEX was used to identify blotted antigen (SC) by adding ACA diluted 1:200 in PBS/5% milk in combination with a Chemiluminescent Substrate Reagent Kit (Invitrogen, Cat No. WP20005). Membranes were imaged as above. APEX activity was also measured on membranes blotted with ACA. Membrane lanes were cut and incubated with either TMB or Amplex^®UltraRed, and APEX activity was measured by using the spectrophotometer to read the absorbance at 450 nm and the fluorescence at 595 nm for TMB and Amplex^®UltraRed, respectively.

Total protein was collected by using RIPA lysis buffer supplemented with protease inhibitor cocktail tablet and phosphatase inhibitors (Roche, USA). Afterward, cells were centrifuged at 14,500 rpm for 10 min at 4°C. Supernatant was employed for protein loading. Then, the proteins were electro-transferred to a PVDF membrane and blocked using 5% nonfat milk powder. After incubation with anti-IRTKS (ab226344, Abcam) and Goat Anti-Rabbit IgG H&L (ab6721, Abcam), the membrane was developed using the Chemiluminescent Substrate Reagent Kit (Invitrogen).

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).