CPSF6 Antibody 75168S from Cell Signaling Technology

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Nature methods
January 01 2022

Fig 1: Principles of X4 HIV-1 fusion and infection assays in resting CD4+ T cells.Two different types of conditions for HIV-1 challenge were used depending on the scientific question, that is the target protein of interest and the step of the HIV replication cycle under investigation. a, First, to characterize the effect of KOs of either host dependency factors (CXCR4, CD4) or restriction factors (PSGL-1) for HIV binding and entry, we employed the well-established HIV fusion assay without spinoculation. Here, cells were exposed to the X4 HIV-1 BlaM-Vpr inoculum for 4 hours at 37 °C. Cells with either CXCR4 KO or nucleofected with NTC-gRNA were used. Representative dot plots of the flow cytometric detection of the CCF2 substrate cleavage by BlaM in viable cells after virion fusion are shown. As specificity controls, cells were pretreated with either the HIV-1 fusion inhibitor T20 or an anti-CD4 mAb. One representative experiment is shown (n = 7). b, Second, to characterize the role of potential cellular restriction factors (SAMHD1, MX2) or host dependency factors (CPSF6) at post-entry steps of the replication cycle (for example at reverse transcription, nuclear import, integration) we sought to efficiently overcome the natural restriction at virion entry and allow a high-level of virus delivery (see Fig. 3 and Fig. 5). In this context, we applied spinoculation at 650 g for 150 min at 37 °C. To prove that this is optimal for high-level virion delivery into resting CD4+ T cells, we performed a quantitative HIV-1 fusion assay. This spinoculation condition allowed X4 HIV-1 to enter into virtually every resting CD4+ T cell that expresses the co-receptor CXCR4, that is typically around 95% of the cell population. As specificity control, the small molecule inhibitor AMD3100, which blocks CXCR4-dependent HIV-1 fusion, was used. Means ± s.e.m. are shown (n = 4). Source data
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Nature methods
January 01 2022

Fig 2: Phenotypic characterization of various single-gene KOs in polyclonal, resting CD4+ T cells.a,b, FACS density plots of surface-exposed CXCR4 (a) or CD46 (b) from one experiment at three time points (n = 3). c, WES immunoblot for SAMHD1 in cultivated KO or NTC cells. Vinculin was the loading control. One representative experiment is shown (n = 4). d, SDF-1a (CXCL12)-driven chemotaxis of cells 1 week after nucleofection. Mean ± s.e.m. are shown (n = 3). Statistics indicate significance by two-way ANOVA. P values were corrected for multiple comparison (Tukey) (***P = 0.0007; **P = 0.0010). e, The frequency of cells (NTC or indicated KOs) positive for cleaved CCF2 substrate after challenge with HIV-1 carrying BlaM-Vpr, indicative of HIV-1 fusion, was determined by flow cytometry. Mean ± s.e.m. are shown (n = 7). Statistics indicate significance by one-way ANOVA. P values were corrected for multiple comparisons (Dunnet) (***P = 0.0002). f, Cells with the indicated KOs were challenged 2 weeks after nucleofection with HIV-1 GFP at two MOIs and analyzed 3 d later. Mean ± s.e.m. are shown (n = 4). Statistics indicate significance by two-way ANOVA. P values were corrected for multiple comparisons (Tukey) (***P = = 0.001). g, Cells were challenged 2 weeks after nucleofection with measles reporter virus MeV-vac-eGFP and analyzed for reporter expression by flow cytometry 1 d later. Means of technical duplicates are shown (n = 2). h, At the indicated weeks after nucleofection, SAMHD1 KO cells or NTC control cells were challenged with HIV-1* GFP either without Vpx (top) or carrying Vpx (+Vpx, bottom) and analyzed 3 d later by flow cytometry. Infection values for the NTC control were set to 1. Mean ± s.e.m. are shown (n = 5). Statistics indicate significance by two-tailed Mann–Whitney U-test. (2 weeks, *P = 0.0286; 4 weeks, *P = 0.0286; 6 weeks, **P = 0.0079). i, Immunoblots for MX2 (top) or CPSF6 (bottom) in cell lysates 4 weeks after nucleofection. Vinculin was the loading control. Two representative donors are shown (n = 4). Ø, empty lane. j,k, HIV-1 fusion (j) and HIV-1 GFP infection (k) in cells with the indicated KOs 4 weeks after nucleofection (as in e and f, respectively). Mean ± s.e.m. are shown (n = 3). Statistics indicate significance by two-way ANOVA. P values were corrected for multiple comparison (Dunnet) (**P = 0.0054).Source data

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CPSF6 Antibody

Cell Signaling Technology's CPSF6 Antibody is a Rabbit Polyclonal antibody. This antibody has been shown to work in applications such as: Precipitation, Immunoprecipitation, and Western Blot. The CPSF6 Antibody was generated using CPSF6 as the antigen and it reacts with Homo Sapiens, Human, Mouse, Rat, and Non-Human Primate.

Description

CPSF6 Antibody recognizes endogenous levels of total CPSF6 protein

Nature methods

Nature methods

CPSF6 Antibody from Cell Signaling Technology

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Cell Signaling Technology

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CPSF6 Antibody from Cell Signaling Technology

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