Journal: Cell and tissue research

Article Title: Modifications of the 26S proteasome during boar sperm capacitation

doi: 10.1007/s00441-017-2786-6

Figure Lengend Snippet: (a) Flow cytometric measurements of boar spermatozoa, after each treatment step, before and after capacitation under proteasomal activity permissive/inhibiting conditions, including vehicle control. Spermatozoa were labelled with an antibody against a shared domain of 20S proteasomal core subunits PMSA1–7) to screen for proteasome modifications, and lectin PNA for outer acrosomal membrane integrity screening. DNA was stained with DAPI. Representative fluorescence images of spermatozoa with double positive labelling of 20S core and OAM (PNA lectin labelling) are shown in a FlowSight image gallery. (a’) Scatted plot representation of 20S and PNA double-labelled spermatozoa, representative of each treatment step, before and after capacitation with/without proteasomal inhibitors, including vehicle control. Each scatter plot represents 10,000 properly focused single cell flow cytometric events. The experiment was replicated twice with comparable results.

Article Snippet: Primary antibodies used for 26S proteasome subunits detection were anti-PMSA1–7 subunits of 20S core subunit (1:200 dilution; BML-PW8155, Enzo), and anti-PSMD4 subunit of 19S regulatory particle (1:200 dilution; ab20239, Abcam).

Techniques: Activity Assay, Staining, Fluorescence

Journal: Cell and tissue research

Article Title: Modifications of the 26S proteasome during boar sperm capacitation

doi: 10.1007/s00441-017-2786-6

Figure Lengend Snippet: (a) Western blot of 20S core subunits PMSA1–7 in sequentially isolated proteins from ejaculated and capacitated spermatozoa, with/without proteasomal inhibitors, and vehicle control. (a’) PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane. (a”) Residual gel after electro-transfer, for protein normalization purposes. Proteins were resolved on 8–20% gradient gel under non-reducing conditions, and protein equivalent of 20 million spermatozoa was loaded per single lane. The experiment was replicated twice with comparable results.

Article Snippet: Primary antibodies used for 26S proteasome subunits detection were anti-PMSA1–7 subunits of 20S core subunit (1:200 dilution; BML-PW8155, Enzo), and anti-PSMD4 subunit of 19S regulatory particle (1:200 dilution; ab20239, Abcam).

Techniques: Western Blot, Isolation, Staining