Gtx125953

HEK293T transfected transiently were treated with 5 μM MG132 for 14 h and washed twice with PBS containing 0.05% N-ethylmaleimide (NEM). Cells were then lysed at 95˚C for 15 min with denaturing IP buffer, which contained a 1:3 ratio of buffer I (5% SDS, 150 mM Tris-HCl at pH 6.7, and 30% glycerol) to buffer II (0.1% SDS, 150 mM Tris-HCl at pH 8.2, 5 mM NaCl, 0.5% NP-40, and 0.1% sodium azide) [39] (link)[40] (link)[41] (link). Proteins in the cell lysate were immunoprecipitated with anti-HA agarose (Sigma, A2095) and immunoblotted according to the methods described above. To detect endogenous polyUb-conjugated NS2, H1299 cells were transfected with pV5-Ub. At 24 h after transfection, cells were infected with A/Puerto Rico/8/ 34 (H1N1) strain at an MOI of 2 for 0, 6, 9, 12, and 18 h. A lysate was prepared with denaturing IP buffer as described above followed by immunoprecipitation overnight at 4˚C with anti-NS2 antibodies (GTX125953, GeneTex) and Protein G agarose beads (Millipore). Immunoprecipitated proteins were detected by immunoblotting according to the methods described above.