我们要去研究USP38对于TBK1的分子机制。
恩，如何研究呢？
In 293T cells transfected with FLAG-tagged TBK1 and HA-tagged IRF3, together with increasing doses of USP38, we found that the concentration of TBK1 proteinde creased considerably with increasing USP38 expression. However, the mRNA level of TBK1 remained unchanged (Figure 4A), suggesting that USP38 causes TBK1 protein degradation.
仔细读一下不难发现TBK1随USP38的增多而减少，但是TBK1的mRNA却不变，暗示了USP38导致TBK1降解。
To determine the specificity of the USP38-mediated TBK1degradation, we used other USP family members USP3 andUSP13 as a control, and we found that USP38, but not USP3or USP13, induced TBK1 degradation (Figure S3A).
这里用了USP3 andUSP13作为对照，证明只有USP38导致TBK1降解。
To determine whether USP38 causes the degradation of other proteins,weperformed a similar experiment and found that USP38 specifically degraded TBK1, but not IKKi, IKK-a, or IKK-b(Figure 4B).
这里用IKKi, IKK-a, or IKK-b做对照，说明只有TBK1被USP38控制降解
To determine whether USP38 degrades TBK1 through aubiquitin-protease pathway, we performed experiments in the presence of the proteasome inhibitor MG-132 or 3-Methyladenine (3-MA), and we found that MG-132, but not 3-MA, blocked the degradation of TBK1 (Figure 4C), suggesting that USP38- induced TBK1 degradation is dependent on a proteasome pathway.
这里用了蛋白酶的抑制剂，就是不让蛋白酶有活性，结果TBK1不降解了，说明USP38是通过一种蛋白酶通道达到控制TBK1的效果的。
Murine USP38 (mUSP38) showed similarinhibitory function of human USP38 protein (Figure S3B),suggesting a conserved function of USP38 between mice and humans.
这一小句说小鼠和人类的USP38功能相似，USP38相对保守。
To determine whether USP38 can mediate degradation ofendogenous TBK1 under physiological conditions, we transfected 293T cells with USP38, and we found that endogenousTBK1 protein was unchanged (Figure S3C).
很奇怪的是外源的USP38并没有使TBK1降解。于是问题来了。
We reasoned that USP38 may specifically target the activated form of TBK1 for degradation.
我们的猜测是USP38是在TBK1激活时才起作用。
于是设计实验。
Therefore, we generated an inactive mutant ofTBK1 with a substitution of alanine for the serine at position 172 (S172A), which abrogates its auto-phosphorylation, and then we co-transfected the 293T cells with USP38, WT TBK1, or TBK1 (S172A). Indeed, we found that USP38 could not interact with mutant TBK1 (S172A) and failed to promote it for degradation (Figures 4F and 4G); however, TBK1 constitutive active mutant (S172E) showed an increasing binding ability to USP38 compared to WT TBK1 (Figure 4F).
这里用了一个失活的TBK1，果然USP38不能使TBK1降解了。
Furthermore, we found that USP38 expression reduced TBK1 protein, but not TBK1 mRNA, after VSV infection compared with 293T cells transfected with the empty vector (EV) (Figures 4H andS3D). Conversely,USP38knockdown in THP-1 cells increased endogenous TBK1 protein levels in cells infected with VSVEGFP or transfected with IC poly(I:C), but not in uninfected cells (Figures 4I andS3E).
这里说的是USP38没有使TNK1的mRNA减少，而USP38knockdown只在cells infected with VSVEGFP or transfected with IC poly中使TBK1增多，而在uninfected cells却不行。
Similar results were obtained with WT or Usp38�/�BMMs with VSV or HSV-1 infection (Figure 4J). These results suggest that USP38 specifically degrades the active form (p-TBK1) of TBK1 after viral infection.
嗯，经过这些证明了在病毒感染后USP38特异性的降低TBK1的活性。
问题：1.如何证明USP38是特异性调控TBK1的。
2.如何证明USP38是针对激活的TBK1的。
