V5 tag antibody - #T0057

Fig 1. Ubiquitination and instability of NS2. (A) HEK293T cells were transfected with pHA-NS2. At 24 h post-transfection, cells were treated with DMSO (lane 1) or MG132 (lane 2) for 14 h. Proteins in the lysates were then detected by immunoblotting with anti-HA and anti-α-tubulin antibodies. (B) HEK293T cells were transfected with pcDNA-HA (lane 1) or pHA-NS2 (lane 2), and then treated with MG132. Proteins in the lysate were immunoprecipitated (IP) with anti-HA antibody and immunoblotted (IB) with anti-Ub (upper panel) and anti-HA (lower panel) antibodies (lanes 1–2). (C) H1299 cells were transfected with pV5-Ub. At 24 h post-transfection, cells were infected with A/Puerto Rico/8/1934 (H1N1) at an MOI of 2 for 0 h (lanes 1, 6), 6 h (lanes 2, 7), 9 h (lanes 3, 8), 12 h (lanes 4, 9), and 18 h (lanes 5,10). Proteins in the lysates were immunoprecipitated with anti-NS2 antibody and immunoblotted with anti-V5 and anti-NS2 to determine ubiquitinated NS2 and non-ubiquitinated NS2, respectively (lanes 1–5). (D) HEK293T cells were cotransfected with pHA-NS2 and pcDNA-NV5 (lane 1), pHA-NS2 and pV5-Ub (lane 2), or pHA-NS2 and pV5-Ub(K0) (lane 3). At 24 h after transfection, cells were treated with MG132 for 14 h. Proteins in the lysates were immunoprecipitated (IP) with anti-HA antibody and immunoblotted (IB) with anti-V5 antibody to show ubiquitinated HA-NS2. The input (B, lanes 3–4; C, lanes 6–10; D, lanes 1–3, lower panel) shows the amount of total ubiquitinated proteins (Ub-P) (B), V5-Ub conjugated proteins (V5-Ub-P) (C, D), NS2 (C), HA-NS2 (D), and α-tubulin (B-D) in the lysates as detected by immunoblotting with anti-Ub, anti-V5, anti-NS2, anti-HA, and anti-α-tubulin antibodies, respectively. PUNS2: polyubiquitinated HA-NS2 (B and D) or NS2 (C). “–” represents an empty vector, pcDNA-HA (A, B) and pcDNA-NV5 (D). (E-H) HEK293T cells were cotransfected with (E) pHA-NS2 and pcDNA-NV5, (F) pHA-NS2 and pV5-Ub, or (G) pHA-NS2 and pV5-Ub(K0). At 36 h post-transfection, cells were treated with cycloheximide (CHX). Proteins in the lysate were detected by immunoblotting with anti-HA, anti-V5, and anti-α-tubulin antibodies at 0, 30, 60, and 120 min after the treatment. (H) Intensity of the HA-NS2 bands in (E-G) was measured with ImageJ and normalized to the intensity of the respective control α-tubulin band. The amount of NS2 at Hour 0 was set to 1. Error bars represent standard error, which was calculated from the results of three independent sets of experiments. *p < 0.05.