Anti ha h6908

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).

For co-immunoprecipitation (Co-IP) assays, HEK293T cells were transfected with Myc-SNAI2 and/or HA-DEAR1 plasmids in a 6 cm plate using Mirus TransIT-LT1 transfection reagent (Mirus, MIR 2300) for 24 h. Cells were treated with MG-132 proteasome inhibitor (Calbiochem, 133407-82-6) for 2 h, before harvest. Cells were lysed with M-PER lysis buffer (ThermoFisher Scientific, 78501) and incubated with pull-down antibody overnight at 4 °C. Then, lysates were incubated with protein A/G agarose beads (Santa Cruz, sc-2003) for 2 h at 4 °C followed by three 5-min washes with RIPA lysis buffer. Proteins were eluted from beads in 30 µL of 2× SDS sample buffer for western blot analysis. Rabbit polyclonal anti-HA (H6908) or anti-Myc (M4439) from Sigma were used for pull-down and Mouse monoclonal anti-HA (H3663) from Sigma or anti-Myc (#562) from MBL International Corporation were used for immunoblotting. For in vivo ubiquitination assays, HEK293T cells were transfected with Myc-SNAI2, 8x-HA-Ub, and/or DEAR1 plasmids in a 10 cm plate. At 20 h after transfection, cells were treated with MG132 20 μmol/L for 4 h and harvested. Cells were lysed with 1× SDS-lysis buffer, denatured by heating at 95 °C for 10 min and then diluted with 0.5% NP-40 buffer and used for further analysis.