Immobilin p

Sperm proteins were solubilized in electrophoresis sample buffer (2% SDS, 10% glycerol, 50 mM DTT, 62.5 mM Tris-HCl, pH 6.8) and heated at 100°C for 5 min, resolved by SDS-PAGE [46 (link)] and transferred onto PVDF membrane (Immobilin-P; Millipore, Bedford, MN, USA) [47 (link)]. Non-specific sites on the membrane were blocked with 5% (w/v) skimmed milk in Tris-buffered saline containing Tween 20 (TBS-T; 150 mM NaCl, 20 mM Tris-HCl, 0.1% Tween 20, pH 7.4). The membrane was next incubated with a monoclonal anti-PDE10A antibody (clone 1C9, recommended by the manufacturer for western blot; Origene Technologies, Rockville, MD) for 16 h at 4°C. Following several washes, the membrane was incubated with a goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase for 1 h (GE Healthcare Bio-Sciences Inc., Baie d’Urfé, QC, Canada or Jackson Immunoresearch, West Grove, PA). After five washes in TBS-T, positive immunoreactive bands were detected using an enhance chemiluminescence kit (ECL Prime; GE Healthcare Bio-Sciences Inc.) according to the manufacturer’s instructions and film exposure (Fuji, Tokyo, Japan), or with ECL clarity chemiluminescence kits (Bio-Rad, Mississauga, ON, Canada) and then scanned with the ChemiDoc MP Imaging System (Bio-Rad).

Purified MERS-CoV VLPs were transferred onto a Polyvinylidene fluoride (PVDF) membrane (Immobilin-P, Millipore, USA) after SDS-PAGE under denaturing conditions for Western blotting with anti-S, E, M mouse polyclonal antibodies.

Western Blot analysis was performed according to our previous study [22] (link). Total cellular proteins were extracted and separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Immobilin-P; Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight with rabbit anti-FZD7 (Santa Cruz, CA, USA), mouse anti-Myc (Santa Cruz, CA, USA), mouse anti-Cyclin D1 (Santa Cruz, CA, USA), mouse anti-β-catenin (Santa Cruz, CA, USA) or mouse anti-β-actin (Sigma, St. Louis, MO, USA) antibodies at 4°C. After incubation with horseradish peroxidase–conjugated anti-rabbit IgG or anti-mouse IgG (Santa Cruz, CA, USA), the specific protein band was visualized by enhanced chemiluminescence (Amersham-Pharmacia Biotech, Beijing, China). β-actin was used as an internal control, and each experiment was repeated at least thrice.

The whole-cell extracts were prepared by washing cells twice with ice-cold 1× PBS and lysed in lysis buffer with the addition of Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktails and PMSF (Kangchen bio-tech, China) for 30 min at 4 °C on a rocking platform before scraping and transferring to tubes. The lysates were cleared by centrifugation at 13,000 g for 20 min at 4 °C, and protein quantification was performed with the BCA protein assay kit (Beyotime, China). For western blotting, 20 μg total protein were prepared with 5× SDS sample buffer and resolved by 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane (ImmobilinP, Millipore) using standard procedures. After blocking in 5% skim milk in Tris-buffered saline–Tween (TBST), incubation for overnight at 4 °C with primary antibodies against GAPDH, β-Tubulin, Sp1, Sp3 (Santa Cruz, USA), GATA1 and STING (Abcam, UK) and then with HRP-conjugated secondary antibodies (Abcam, UK) was performed. Subsequently, Chemoluminescense signals of membranes were quantified with ECL reagent (Pierce, USA) and visualized by enhanced chemiluminescence (Cell Signaling Technology, Inc). Anti-GAPDH or β-Tubulin was used as loading control.