我們要去研究USP38對(duì)于TBK1的分子機(jī)制傻咖。
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.
仔細(xì)讀一下不難發(fā)現(xiàn)TBK1隨USP38的增多而減少，但是TBK1的mRNA卻不變府框，暗示了USP38導(dǎo)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).
這里用了USP3 andUSP13作為對(duì)照讥邻，證明只有USP38導(dǎo)致TBK1降解迫靖。
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).
這里用IKKi, IKK-a, or IKK-b做對(duì)照，說(shuō)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.
這里用了蛋白酶的抑制劑兴使，就是不讓蛋白酶有活性系宜，結(jié)果TBK1不降解了，說(shuō)明USP38是通過(guò)一種蛋白酶通道達(dá)到控制TBK1的效果的发魄。
Murine USP38 (mUSP38) showed similarinhibitory function of human USP38 protein (Figure S3B),suggesting a conserved function of USP38 between mice and humans.
這一小句說(shuō)小鼠和人類(lèi)的USP38功能相似盹牧，USP38相對(duì)保守俩垃。
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并沒(méi)有使TBK1降解。于是問(wèn)題來(lái)了汰寓。
We reasoned that USP38 may specifically target the activated form of TBK1 for degradation.
我們的猜測(cè)是USP38是在TBK1激活時(shí)才起作用口柳。
??????????????????????????????????????????????????????????????????? 于是設(shè)計(jì)實(shí)驗(yàn)。
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).
這里用了一個(gè)失活的TBK1有滑，果然USP38不能使TBK1降解了跃闹。
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).
這里說(shuō)的是USP38沒(méi)有使TNK1的mRNA減少，而USP38knockdown只在cells infected with VSVEGFP or transfected with IC poly中使TBK1增多俺孙，而在uninfected cells卻不行辣卒。
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.
嗯掷贾，經(jīng)過(guò)這些證明了在病毒感染后USP38特異性的降低TBK1的活性睛榄。
問(wèn)題：1.如何證明USP38是特異性調(diào)控TBK1的。
?????????? 2.如何證明USP38是針對(duì)激活的TBK1的想帅。
