Briefly, 96-wells bottom polystyrene microplates were coated with cell lysates (50 μl/well) and incubated for 1 h at 37 °C and then washed by washing buffer before incubated with blocking buffer for 90 min at 37 °C.
The cells were re-suspended in Annexin-V binding buffer (BD, Oxford, UK) before incubating with monoclonal antibody Annexin-V conjugated with Alexa 488 (10 μg/ml) (Sigma, UK) for 30 min avoiding light.
Sections were fixed in 10% NBF for 20 minutes at room temperature and washed with phosphate buffered saline (PBS) before incubating with 10 μg/ml of conjugated agglutinin while gently rocking in darkness for 2 hours at room temperature.
GST-Slx9 or GST-Ssb1C was immobilized on GSH-Sepharose before incubating with either buffer alone or buffer containing 2 µM His6-RanQLGTP, 50 nM Crm1-His6 or 2 µM His6-RanQLGTP, and 50 nM Crm1-His6.
To confirm equal loading, membranes were immersed in stripping buffer at 50 °C for 30 min before incubating with 1 5000 anti-β-actin (A1978; Sigma, Poole, United Kingdom) and the antimouse sc-23711; secondaryz) secondary.
Before incubating with cell lysates, membranes were incubated with blocking buffer for 1 h at room temperature.
Before incubating with antibody, the membrane was blocked in blocking buffer (1 × phosphate-buffered saline (PBS), 0.1% Tween-20, 5% skim milk) for 30 min at RT.
with 0.1% Tween-20/PBS Tween-20/PBSating with primary antibefore.
The inner ears were washed three times with PBS buffer before incubating in PBS containing 0.25 M EDTA for 2 days at 4°C.
For the PLZF FACS, the P9 testes cell suspensions were first fixed and washed using the transcription factor buffer set (BD Biosciences, 562574) before incubated with PLZF (D-9) (0.2 μg/test) (sc-28319), then the Cy5-conjugated Goat-anti mouse antibody (Jackson ImmunoResearch Laboratories, 715-175-150 (0.5 μg/ml, 1 : 200)) were incubated after washing by wash/perm provided in the buffer set.
