Figure 11.

Phenotyping of STK39 in non-capacitated (NC) and in vitro capacitated (IVC) spermatozoa with and without proteasomal inhibition, including a vehicle control. ICC detection of STK39 (red) in NC (a) and IVC, non-inhibited (a’) spermatozoa; the inset shows STK39 channel only. Spermatozoa were co-stained for acrosomal integrity with peanut agglutinin (PNA lectin, green) and a nuclear stain DAPI (blue). All fluorescence channels were superimposed with the DIC brightfield channel. Scale bars represent 20 µm. The IBFC of formaldehyde fixed and Triton-X-100-permeabilized NC and IVC spermatozoa with or without proteasomal inhibition, including a vehicle control and negative immunolabeling control, normal rabbit serum (b). Immunofluorescence images of NC (b’) and IVC spermatozoa (b”) obtained by using IBFC to confirm the labeling. The IBFC was performed in four replicates with consistent results. Immunoprecipitation of STK39 and WB detection (c) in the extracts (input), immunoprecipitates (IP), and non-binding (flow-through, FT) fractions obtained from NC and IVC spermatozoa. The upper red arrow points to the expected STK39 band (predicted molar weight of 63 kDa) and the lower arrow point to the assumed, processed form after the sperm in vitro capacitation. The bands at ~53 kDa and 25 kDa were from the heavy and light chains, respectively, of the STK39 antibody, as anticipated. The membrane was stained with CBB R-250 (c’) for protein load estimation. Statistical evaluation of the IBFC results (b’’’) was performed by using ANOVA with Sidak’s post hoc test (α = 0.05).