Anti myc 9e10

Cells were washed with ice-cold PBS and lysed on ice for 30 min in RIPA lysis buffer (150 mM NaCl, 5% Glycerin, 1% Triton X-100, 50 mM Tris-HCl, pH7.5). For each immunoprecipitation reaction, 0.2 ml of the cell lysate was incubated with 1 μg of antibody (anti-Myc, 9E10, Santa Cruz, sc-40; anti-HA, H6908, Sigma-Aldrich) at 4 °C for 4 h, and then incubated with 10 μl of protein A/G agarose beads at 4 °C for 1 h. The sediments were then subjected to SDS-PAGE and immunoblot analysis. The antibodies used in immunoblotting were as follows: anti-Myc (9E10, Santa Cruz,1:2000), anti-HA (H6908, Sigma-Aldrich, 1:2000), anti-Flag (F3165, Sigma-Aldrich, 1:15000), anti-LC3B (ab192890, Abcam,1:1000), anti-IFITM3 (11714-1-AP, Proteintech,1:5000), anti-STAT1 (14994T, Cell Signaling Technology, 1:1000), anti-pSTAT1 (Tyr701) (7649T, Cell Signaling Technology, 1:1000), anti-FOXP3 (14-7979-82, eBioscience, 1:3000), anti-FOXO1 (2880T, Cell Signaling Technology, 1:1000), anti-Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) (9464T, Cell Signaling Technology, 1:1000), anti-AKT (60203-2-Ig, Proteintech, 1:5000), anti-p-AKT (9271T, Cell Signaling Technology, 1:1000), anti-GAPDH (60004-1-Ig, Proteintech, 1:8000), anti-β-Actin (66009-1-1g, Proteintech, 1:8000), anti-LaminB (66095-1-Ig, Proteintech, 1:8000).

Testes were collected from 1–3-day-old males as previously described (Zamore and Ma, 2011 (link)). Testes were snipped at level 2 and immunostained as previously described (Sitaram et al., 2014 (link)). Anti-Myc (9E10, 1:100; Santa Cruz Biotechnology, catalog #sc-40) and anti-tubulin (DM1α, 1:200; Sigma-Aldrich, catalog #T6199) primary antibodies were used in combination with goat anti-mouse Cy2 secondary (1:400; Invitrogen, catalog #A-11004).
Localization of Myc-tagged Cdc14 protein in 0–2 embryos laid by females carrying the UASp-cdc14-myc and nanos-Gal4 transgenes was assessed by immunofluorescence using standard conditions. Anti-Myc (9E10, 1:100; Santa Cruz Biotechnology, catalog #sc-40) and rat anti-alpha tubulin (1:200; Accurate Chemical & Scientific, Westbury, USA, catalog #MCA77G) primary antibodies were used in combination with goat anti-mouse Cy2 (1:400; Invitrogen, catalog #A-11004) and goat anti-rat Cy3 (1:400; Abcam, catalog #ab6953) secondary antibodies.

For immunoprecipitation experiments, cell lysates were prepared with lysis buffer A and precleared with protein G-Sepharose beads (GE Healthcare) at 4 °C for 1 h. Precleared lysates were incubated with an appropriate antibody [anti-Flag M2 (Sigma, F1804; 3 µg/sample) or anti-Myc 9E10 (Santa Cruz, sc-40; 1 µg/sample) antibody] for 3 h with gentle rotation. protein G-Sepharose beads were then added and incubated for 1 h. Immunoprecipitates were collected by centrifugation and washed three times with lysis buffer A containing 0.1% SDS and subjected to SDS-PAGE. For co-immunoprecipitation assays, cells were harvested with lysis buffer B and sonicated using Qsonica Q700 (QSonica) and centrifuged. The supernatants were incubated with an appropriate antibody [anti-Myc 9E10 antibody (Santa Cruz, sc-40; 1 µg/sample) or mouse control IgG₁ G3A1 (CST, 5415; 1 µg/sample)] overnight at 4 °C. Protein G-Dynabeads (Thermo Fisher) were then added and incubated for 1 h. Immunoprecipitates were washed three times with lysis buffer B. Proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies.