Fig. 5
Phenotyping of RAB2A protein in the non-capacitated (NC) and in vitro capacitated (IVC) spermatozoa with and without proteasomal inhibition, including vehicle control. The ICC detection of RAB2A (red) in NC (a1) and IVC, non-inhibited (a2) spermatozoa, including non-immune serum control (a3). Spermatozoa were co-stained for acrosomal presence with peanut agglutinin (PNA, green), and nuclear stain DAPI (blue). All fluorescence channels are superimposed with the differential interference contrast (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 and negative, normal rabbit serum control (b1). Immunofluorescence images of NC (b2) and IVC spermatozoa (b3), including normal mouse serum (b4) obtained by IBFC to confirm labeling. The IBFC was performed in 4 replicates with consistent results. Detection of RAB2A by WB (c1) in the extracts obtained from NC and IVC spermatozoa under proteasome permissive and inhibiting conditions including vehicle control. The red arrows point to the 24 and 27 kDa RAB2A band doublet reported previously80, and the blue arrow points to the 31 kDa form observed here for the first time. The membrane was stripped and reprobed with anti-TUBB antibody (c2) and stained with CBB R-250 (c3) for protein load normalization purposes. Western blotting was performed in 6 replicates. Statistical evaluation of the IBFC results (b5) and WB results (c4) was performed by using ANOVA with Sidak’s post hoc test (α = 0.05). Full, uncropped membranes of all six replicates of TUBB and RAB2A WB detections are presented in Fig. S7 A and B, respectively. RAB2A blocking peptide assay and Western blotting of recombinant human RAB2A are presented in Fig. S8 and S9, respectively.
