Anti ty1 antibody

Samples were lysed to the concentration of 5.0 × 10⁵ OSC/1 ovary per 10 μl 1× Laemmli buffer and heated at 95°C for 5 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a 0.45 μm nitrocellulose membrane (Cytiva). Membrane was washed by PBS and blocked with 3% skim milk in PBS-T, then incubated with primary antibody for 1 h at room temperature. For primary antibody, anti-beta tubulin (DSHB, E7) (1:5000), anti-c-Myc (DSHB, 9E10) (1:1000), anti-Ty1 antibody (Diagenode, C15200054) (1:2000), anti-HeT-A Gag supernatant (this study) (1:1), anti-Mod(mdg4) common region supernatant (this study) (1:4) and anti-Mod(mdg4) variant N supernatant (this study) (1:1) were used with indicated dilutions. After three washes with PBS-T, membrane was incubated with HRP-conjugated secondary antibody (MP Bioscience, 0855558) (1:5000) for 30 min, followed by three PBS-T washes. The membranes were incubated at room temperature for 1 h. The membrane was incubated with ECL Prime Western Blotting Detection Regent (Cytiva) and was exposed to Amersham Hyperfilm ECL (Cytiva). Exposed film was developed by X-ray film developer (KONICAMINOLTA TCX-101).