Ge10600002

Epididymides from control (n = 3 rats) and experimental animals (castrated and castrated with hormone replacement, n = 3 rats for each group) were processed similarly to those for immunoblotting as described above, with the epididymal fluid being used for this assay. Commercial sepharose beads conjugated with protein A (Amersham Bioscience) were washed twice with 1 ml PBS (phosphate buffered saline) and centrifuged at 3000 x g for 3 min. The beads were resuspended in 100 μl of PBS and incubated with 2 μg of anti-prosaposin antibody for 2 h at 4°C. After incubation, the beads were centrifuged at 3000 x g for 5 min and washed twice with PBS. The beads were resuspended in 100 μl of PBS and incubated for 16 h at 4°C with 1 ml of epididymal fluid after which they were spun down at 3000 x g for 5 min and washed twice with PBS. The proteins bound to the beads were solubilized with Laemmli buffer, analyzed by SDS-PAGE and electrotransferred to nitrocellulose membranes (GE10600002, GE Healthcare, Amersham, Germany). The co-immunoprecipitated anti-cathepsin D was detected by immunoblotting as detailed above. Detection of rabbit IgG (heavy chain) in the precipitates was used as loading control.

Ten micrograms of each sample were electrophoresed through a 12-well, 4–20% SDS–PAGE gel (Bio-Rad #4561096) at 100V for 1 hr at RT, transferred onto a nitrocellulose membrane (GE Life Sciences #GE10600002) at 100 V for 70 min at RT on ice, and blocked with 5% nonfat dry milk in TRIS-buffered saline containing 0.05% Tween-20 (TBST, pH 7.4). Gels were saved for Coomassie staining at RT for 30 min. Membranes were probed with one primary antibody at a time for 24 hr at 4°C and then washed four times with TBST at RT before incubating with the corresponding species-specific secondary antibody at RT for 1 hr. Membranes were washed with TBST, and then enhanced chemiluminescence (ECL) substrate was added (Thermo Fischer #32109). Membranes were exposed to autoradiography films and scanned with an Epson 1670 at 600dpi. We probed each membrane with one antibody at a time, stripped the membrane with stripping buffer (Thermo Fischer #21059) for 10 min at RT, and then blocked for 1 hr at RT before probing with the next antibody. Order of probes: Strumpellin, β-tubulin, and then WASH1. We determined the optical density of the bands using Image J software (NIH). Data obtained from three independent experiments were plotted and statistically analyzed using GraphPad Prism (version 8) software.

Eight micrograms of each sample were electrophoresed through a 15-well, 4–20% SDS–PAGE gel (Bio-Rad #4561096) at 100 V for 1 hr at RT and transferred onto a nitrocellulose membrane (GE Life Sciences #GE10600002) at 100 V for 70 min at RT on ice. Membranes were incubated with Total Protein Stain for 5 min at RT (LI-COR # 926–11015), rinsed, and imaged at 700 nm using an Odyssey Fc imaging system (LI-COR) to determine protein loading. Membranes were then briefly incubated with REVERT solution and blocked with Odyssey blocking buffer (LI-COR #927–50000) for 1 hr at RT. Membranes were probed with one primary antibody at a time for 24 hr at 4°C and then washed four times with 1× TBST before incubating with secondary antibody at RT for 1 hr. Membranes were washed four times with TBST and then imaged with an Odyssey Fc imaging system at 700 nm and 800 nm. Order of probes: LAMP1 then EEA1. We determined the optical density of the bands using Odyssey Fc Image Studio software. Data obtained from three independent experiments and statistically analyzed using GraphPad Prism (version 8) software.