Hpa021624

The protein was separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocked with 2% non-fat milk, the membrane was incubated with primary rabbit polyclonal antibodies against ACTL7A (1:1000; HPA021624, Atlas Antibodies); PAWP (1:1000; 22587-1-AP, Proteintech, Chicago, IL, USA) or Acrosin (1:1000; NBP2-14260; Novus Biologicals, Littleton, CO, USA) at 4°C overnight and then with the horseradish peroxidase–labeled antibodies (1:10 000; CWBiotech, Beijing, China). After washed with TBST (20 mmol l⁻¹ tris-HCl, 150 mmol l⁻¹ NaCl, and 0.05% [v/v] Tween 20), the bands were detected using an ECL kit (GE Amersham, Pittsburgh, PA, USA).7 (link)

Immunofluorescence was performed as previously described.7 (link)13 (link)14 (link) Simply, for immunofluorescence, the samples were prepared as smears. The smears were permeabilized with 0.2% Triton X-100 for 30 min, blocked with 30% donkey serum for 1 h, and incubated with ACTL7A (1:50; HPA021624, Atlas Antibodies, Stockholm, Sweden) at 4°C overnight. On the 2^nd day, the slides incubated with donkey anti-rabbit conjugated with Alexa Fluor 488 (1:300; A32731, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature and observed under Nikon A1+ Confocal Microscope System (Nikon, Tokyo, Japan).