Anti smurf2

Western blot analysis was performed as previously described (Park et al., 2012 (link)). Proteins were isolated from cells or mouse tissues by homogenization in RIPA buffer with complete mini protease inhibitor cocktail (Roche). Protein concentrations were determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific). Anti-IKKβ, anti-IKKα, anti-Smurf2, and anti-Histone H3 antibodies were purchased from Cell Signaling Technology; anti–β-catenin, anti–β-actin, and anti-GAPDH antibodies were purchased from Sigma-Aldrich; and antiubiquitin monoclonal antibody was purchased from Santa Cruz Biotechnology. For immunoprecipitation experiments, control or shIKKβ 3T3-L1 cells, or adipose SV cells were incubated with 100 nM of PS-341 for 4 h. The whole-cell lysates were isolated, incubated with anti–β-catenin antibody overnight at 4°C, and then incubated with Protein A Agarose beads (Roche) for another 5 h. The samples were washed and analyzed by Western blot using antiubiquitin monoclonal antibodies.

HEK293T cells were transfected with FLAG‐labelled Smurf2, Myc‐labelled HIPK2, HA‐labelled Ubiquitination using Lipofectamine 3,000, followed by lysing in lysis buffer. M2 magnetic beads (catalogue number: A4596, Sigma‐Aldrich) were adopted for immunoprecipitation at 4°C overnight. After washing for four times, the conjugated proteins were detected by western blotting with primary antibodies against Smurf2, HIPK2, Flag, Myc, and HA.
For another experiment, the HL‐1 cell lysates (500 μg) were immunoprecipitated with anti‐IgG (#3900, Cell Signaling Technology) or anti‐Smurf2 (#12024, Cell Signaling Technology) and incubated with protein G‐Agarose beads (Yeasen, Shanghai, China) at 4°C overnight. Subsequently, washing with 1 mL lysis buffer was performed for four times. Finally, the samples were subjected to western blotting. Three independent experiments were conducted.