Ap156p

Proteins were extracted from cell lines using mammalian protein extraction reagent (MPER; 50 nM Tris-HCl, 200 mM NaCl, 0.25% Triton 100X, and 10% glycerol) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, PIA32961). Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, 500-0006). Twenty micrograms of total protein extract were separated in precast 4–15% gradient Tris-glycine SDS-polyacrylamide gels (Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad, 456-1086) and transferred onto Trans-Blot Turbo Mini 0.2 µm nitrocellulose membranes (Bio-Rad, 170-4159). Membranes were blocked with 5% milk in PBS-Tween for 1 h and probed with primary antibodies overnight at 4 °C with agitation. Primary antibodies were detected with peroxidase-conjugated secondary antibodies Goat anti-mouse (Millipore, AP124P, 1:4000 dilution), Goat anti-rabbit (Millipore, AP156P, 1:10,000 dilution), and Rabbit anti-goat (Millipore, AP106P, 1/4000 dilution) and enhanced with chemiluminescence (Millipore, RPN2232) detected using the ChemiDoc MP Imaging System (Bio-Rad). Actin was used as a loading protein control. Each experiment was repeated three times. Image J was used to quantify western blot.

Protein extraction and western blotting was performed using standard procedures. In brief, 30 μg protein per sample was separated by Bolt^® Bis‐Tris 4–12% SDS/PAGE (Thermo Fisher) and blotted onto polyvinylidene fluoride (PVDF) by iBlot^® (Invitrogen, Carlsbad, CA, USA) and blocked by 4% skim milk in 2% Tris‐buffered saline/Tween. Primary antibodies used in this study were as follows: 2A peptide 1 : 2000 ABS31 (Millipore, Billerica, MA, USA), SV40LT 1 : 400 Pab416 (Abcam, Cambridge, UK), and β‐actin 1 : 10 000 AC‐15 (Abcam). Secondary horseradish peroxidase (HRP) antibodies used in this study were as follows: goat anti‐rabbit (AP156P; Millipore) and rabbit anti‐mouse (ab97046; Abcam).

Three-fold dilution series of purified recHPL and PPL (each 100 μl) were dot-blotted onto polyvinylidene fluoride (PVDF) membranes and blocked with 3% BSA in 10 mM Tris-HCl saline (pH 7.5), then cut into lanes and reacted with the rabbit anti-HPL IgGs for immunoblotting (Abcam, ab96100, 1:1,000) and the HRP labelled anti-rabbit IgG Fc (Millipore, AP156P, 1:5,000) at room temperature for 1 h each. The spots were visualized by ECL reagent (GE Healthcare ECLTM Western Blotting Detection Reagents). Chemiluminescence was detected using an ImageQuant LAS4000 mini imager (GE Healthcare). The plotted signal intensities were calculated using ImageJ (NIH) software.