rabbit anti ddx3x antibody  (Proteintech)

Journal: bioRxiv

Article Title: DEAD-box RNA helicase DDX3X maintains the homeostasis of the Zika virus translation-replication cycle

doi: 10.64898/2026.03.27.714787

Figure Lengend Snippet: (a) Intracellular total viral RNA detection by RT-qPCR at 24 hpi at MOI 3 in C20, A549 and HEK293T. 18S ribosomal RNA was used as housekeeping gene. Graph was truncated in Y axis. (b) Western blot of ZIKV infection in C20, A549 and HEK293T cells. Viral protease NS3, DDX3X and 𝛽-actin were evaluated at 24 hpi at MOI 3. (c) Viral titers in supernatant of DDX3X knockdown in C20 (left), A549 (middle) and HEK293T (right) cells determined by plaque assay. (d) Intracellular total viral RNA detection by RT-qPCR at 12, 24, 36 and 48 hpi at MOI 3 in human microglia cells. 18S ribosomal RNA was used as housekeeping gene. (e) Western blot of infection kinetics in C20 cells. Viral protease NS3, viral polymerase NS5, DDX3X and 𝛽-actin were evaluated at 12, 24, 36 and 48 hpi at MOI 3. Right panel, densitometric quantification of NS3 signal. (f) Densitometric quantification of DDX3X protein signal during ZIKV-infection kinetics in human microglia. (g) DDX3X mRNA detection by RT-qPCR at 12, 24, 36 and 48 hpi at MOI 3. 18S ribosomal RNA was used as housekeeping gene. (h) IFN-β mRNA detection by RT-qPCR at 24 hpi at MOI 3 in the three cells lines. 18S ribosomal RNA was used as housekeeping gene. Data are mean ± SEM from three independent experiments. Statistical significance was determined by One-way ANOVA and p -values are indicated in the plot.

Article Snippet: Silencing efficiency was evaluated by Western blot using anti-DDX3X antibody (Cat. 11115-1-AP, Proteintech).

Techniques: RNA Detection, Quantitative RT-PCR, Western Blot, Infection, Knockdown, Plaque Assay

Journal: bioRxiv

Article Title: DEAD-box RNA helicase DDX3X maintains the homeostasis of the Zika virus translation-replication cycle

doi: 10.64898/2026.03.27.714787

Figure Lengend Snippet: (a) Confocal microscopy of infected C20 cells at 24 hpi at MOI 3. DDX3X in green, viral protease NS3 in red and nuclei was stained with DAPI. Inset correspond to the summatory of Z-stack reconstruction of colocalization volume using Imaris software. Right panel corresponds to Mander’s Overlap Coefficient (M) between NS3 and DDX3X signals. M1 of NS3 and DDX3X = 0.4 and M2 of DDX3X and NS3 = 0.247. (b) Western blot of DDX3X-knockdown C20 cells infected with ZIKV at 24 hpi. Viral polymerase NS5, viral protease NS3, DDX3X and 𝛽-actin were evaluated. Right panels correspond to densitometric quantification of NS3 and NS5 protein signals in DDX3X-knockdown conditions in C20 cells. (c) RNA fluorescent in situ hybridization coupled to indirect immunofluorescent microscopy (RNA FISH-IFI) was carried out in C20 cells infected for 24 hpi. Viral genome was detected using a digoxigenin-labeled RNA probe complementary to viral genome (in red), DDX3X in green and nuclei was stained with DAPI. Inset correspond to the summatory of Z-stack reconstruction of colocalization volume using Imaris software. The right panel correspond to Mander’s Overlap Coefficient (M) between vRNA and DDX3X signals. M1 of vRNA and DDX3X = 0.223 and M2 of DDX3X and vRNA = 0.264. (d) Western blot of DENV-2-infected C20 cells at 24 hpi. NS3, DDX3X and 𝛽-actin were evaluated. Right panel correspond to densitometric quantification of DDX3X protein signal in DENV-2-infected C20 cells. (e) Western blot of DDX3X-knockdown C20 cells infected with DENV-2 at 24 hpi. Viral protease NS3, DDX3X and 𝛽-actin were evaluated. Right panel correspond to densitometric quantification of NS3 protein signal in DDX3X-knockdown infection conditions. Data are mean ± SEM from three independent experiments. Statistical significance was determined by t-test, p -values are indicated in the plot.

Article Snippet: Silencing efficiency was evaluated by Western blot using anti-DDX3X antibody (Cat. 11115-1-AP, Proteintech).

Techniques: Confocal Microscopy, Infection, Staining, Software, Western Blot, Knockdown, In Situ Hybridization, Microscopy, Labeling

Journal: bioRxiv

Article Title: DEAD-box RNA helicase DDX3X maintains the homeostasis of the Zika virus translation-replication cycle

doi: 10.64898/2026.03.27.714787

Figure Lengend Snippet: (a) Electrophoretic mobility shift assay to evaluate DDX3X (132–607) and ZIKV 5’UTR interaction. Constant RNA concentration (100 nM) was incubated with increasing DDX3X (132–607) concentrations (0 to 15 μM). (b) Three-dimensional representation of ZIKV 5’UTR by simulation via HDOCK server. Conserved flaviviral functional regions are designated as follows: stem-loop A (gray), stem-loop B (red), capsid hairpin (cHP, yellow), and capsid coding region 1 (CCR1, purple). ZIKV 5’UTR sequence obtained from ZIKV isolate BeH819015 (GI: 975885966) was used. (c) Computational simulation of DDX3X-5’UTR interaction using HDOCK server. Representative structure of the last simulation frame with a close-up of the most relevant interactions during trajectory. Below, inset of DDX3X and 5’UTR principal interactions. Each DDX3X monomer was colored as follow: monomer A, with its N-terminal domain 1 (NT1-D1) in light blue and domain 2 at C-terminal end (D2-CTE) in blue. For monomer B, NT1-D1 in orange, and D2-CTE in yellow. (d) Summary of interactions between DDX3X and 5’UTR in the last frame of simulation using NUCPLOT software. (e) Cell-free translation in RRL was monitored using the viral reporter RNA and mRNA control incubated with increasing DDX3X:RNA molar ratio. Data are mean ± SEM from three independent experiments. Statistical significance was determined by One-way ANOVA compared against respective RNA reporter and t-test. p -values are indicated in the plot.

Article Snippet: Silencing efficiency was evaluated by Western blot using anti-DDX3X antibody (Cat. 11115-1-AP, Proteintech).

Techniques: Electrophoretic Mobility Shift Assay, Concentration Assay, Incubation, Functional Assay, Sequencing, Software, Control

Journal: bioRxiv

Article Title: DEAD-box RNA helicase DDX3X maintains the homeostasis of the Zika virus translation-replication cycle

doi: 10.64898/2026.03.27.714787

Figure Lengend Snippet: (a) Total viral RNA (vRNA) detection by RT-qPCR in DDX3X-knockdown conditions in C20 cells. 18S ribosomal RNA was used as housekeeping gene. (b) RNA FISH-IFI was carried out in DDX3X-knockdown C20 cells infected for 24 hpi. Viral genome was detected using a digoxigenin-labeled RNA probe (in red), DDX3X in green and nuclei was stained with DAPI. The right panels correspond to quantification of mean fluorescence intensity (MFI) of DDX3X and ZIKV gRNA signals in DDX3X knockdown microglia cells. Software ImageJ/Fiji was used. (c) Positive (left) and negative strand (right) viral RNA detection by strand specific RT-qPCR in DDX3X-knockdown conditions. 18S ribosomal RNA was used as housekeeping gene. (d) Confocal microscopy of infected C20 cells at 24 hpi. DDX3X in green, viral polymerase NS5 in red and nuclei was stained with DAPI. Inset correspond to the summatory of Z-stack reconstruction with Imaris software. (e) DDX3X co-immunoprecipitation was performed, viral polymerase NS5 and viral helicase NS3 was evaluated by western blot of immunoprecipitated fractions. (f) Simulation of the interaction between DDX3X-NS5. Disassembly of DDX3X dimer after 15 ns of DDX3X-NS5 system simulation. Stable interaction between DDX3X monomer A (blue) and NS5 (red) remains until the end of simulation. Statistical significance was determined by t-test compared against respective siRNA control group. p -values are indicated in the plot.

Article Snippet: Silencing efficiency was evaluated by Western blot using anti-DDX3X antibody (Cat. 11115-1-AP, Proteintech).

Techniques: Quantitative RT-PCR, Knockdown, Infection, Labeling, Staining, Fluorescence, Software, RNA Detection, Confocal Microscopy, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: DEAD-box RNA helicase DDX3X maintains the homeostasis of the Zika virus translation-replication cycle

doi: 10.64898/2026.03.27.714787

Figure Lengend Snippet: (a) Western blot of ZIKV-infected C20 cells treated with increasing RK-33 concentrations. NS5, NS3, DDX3X and 𝛽-actin were evaluated. The right panels correspond to densitometric quantification of NS3 and NS5 protein signals in RK-33 treatment conditions in C20 infected cells. (b) Total viral RNA (vRNA) detection by RT-qPCR in RK-33 treatment conditions. (c) CLIP-RTqPCR of DDX3X during ZIKV infection in microglia cells treated with DMSO or RK-33 at 3 μM for 24 hours. IgG fraction was used a specificity control of immunoprecipitation and input of DMSO treated cells was used for normalization and comparison. Data are mean ± SEM from three independent experiments. (d) Viral titers in supernatant of C20 infected cells treated with RK-33. Data are mean ± SEM from three independent experiments. Statistical significance was determined by One-way ANOVA and p -values are indicated in the plot.

Article Snippet: Silencing efficiency was evaluated by Western blot using anti-DDX3X antibody (Cat. 11115-1-AP, Proteintech).

Techniques: Western Blot, Infection, Quantitative RT-PCR, Control, Immunoprecipitation, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cell type-dependent suppression of the RNA helicase DDX3Y levels by the close paralog DDX3X

doi: 10.3389/fcell.2026.1644807

Figure Lengend Snippet: U87MG Clone 2 cells generated by CRISPR/Cas9 express reduced DDX3X protein. (A) Diagram of the genotyping results of Clone 2 and the putative encoded DDX3X protein. After the deletion of 14 nucleotides caused by CRISPR/Cas9, which was confirmed at both genomic DNA and cDNA levels, Clone 2 cells likely utilized a downstream translation start site to restore the open reading frame and prevent a complete loss of DDX3X protein. Residues in red were introduced by the mutation, while those in green were unchanged. (B) Representative western blots showing endogenous DDX3X in parental U87MG and Clone 2 cells (three biological replicated each), obtained using anti-DDX3X antibodies from two different sources. Quantification of the DDX3X band intensity detected with the Santa Cruz (SC) antibody is shown on the right. (C) Clone 2 cells were transfected with 100 nM control (CT), DDX3X and 3Y siRNA, and cell lysates were processed for western blotting with the SC antibody.

Article Snippet: Primary antibodies used in this study include mouse anti-DDX3X (Santa Cruz sc-81247; 1:1,000), rabbit anti-DDX3X (Bethyl Laboratories A300-474A; 1:1,000), rabbit anti-DDX3X (Aviva OAAB01241; 1:1,000), rabbit anti-HA (Cell signaling Technology C29F4; 1:2,000), rabbit anti-GFP (Invitrogen A-11122,1:1,000) and custom-made rabbit DDX3Y antibody (1:2,000) ( ).

Techniques: Generated, CRISPR, Mutagenesis, Western Blot, Transfection, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cell type-dependent suppression of the RNA helicase DDX3Y levels by the close paralog DDX3X

doi: 10.3389/fcell.2026.1644807

Figure Lengend Snippet: U87MG Clone 2 cells generated by CRISPR/Cas9 express reduced DDX3X protein. (A) Diagram of the genotyping results of Clone 2 and the putative encoded DDX3X protein. After the deletion of 14 nucleotides caused by CRISPR/Cas9, which was confirmed at both genomic DNA and cDNA levels, Clone 2 cells likely utilized a downstream translation start site to restore the open reading frame and prevent a complete loss of DDX3X protein. Residues in red were introduced by the mutation, while those in green were unchanged. (B) Representative western blots showing endogenous DDX3X in parental U87MG and Clone 2 cells (three biological replicated each), obtained using anti-DDX3X antibodies from two different sources. Quantification of the DDX3X band intensity detected with the Santa Cruz (SC) antibody is shown on the right. (C) Clone 2 cells were transfected with 100 nM control (CT), DDX3X and 3Y siRNA, and cell lysates were processed for western blotting with the SC antibody.

Article Snippet: Primary antibodies used in this study include mouse anti-DDX3X (Santa Cruz sc-81247; 1:1,000), rabbit anti-DDX3X (Bethyl Laboratories A300-474A; 1:1,000), rabbit anti-DDX3X (Aviva OAAB01241; 1:1,000), rabbit anti-HA (Cell signaling Technology C29F4; 1:2,000), rabbit anti-GFP (Invitrogen A-11122,1:1,000) and custom-made rabbit DDX3Y antibody (1:2,000) ( ).

Techniques: Generated, CRISPR, Mutagenesis, Western Blot, Transfection, Control