Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection.

doi: 10.4049/jimmunol.180.4.2396

Figure Lengend Snippet: FIGURE 6. Analysis of signaling proteins in phagosome compartments. MHS cell lysates were processed for phagosome isolation by the sucrose density gradient method without detergent to keep the intracellular mem- branes intact. A, Distribution of key signaling mediators associated with internal vesicles without infection. Clathrin and transferrin receptor (CD71) are markers for receptor-mediated endocytotic vesicular fractions (5–10); caveolin-1 indicates raft-associated fractions (2, 3, 4). B, Phago- some fractions following PAO1 infection were resolved by SDS-PAGE and analyzed by immunoblotting with Lyn, PI3K, Akt, flotillin, LAMP-1, Rab5, Rab7, and PA Abs, respectively. The above results are representative of three experiments.

Article Snippet: Polyclonal rabbit Abs against Lyn, Akt, PI3K, Rab5, Rab7, Flotillin 1, LAMP-1, transferrin receptor, Caveolin-1, and goat polyclonal Abs raised against GAPDH were from Santa Cruz Biotechnology.

Techniques: Isolation, Infection, SDS Page, Western Blot

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion does not affect Rab5 recruitment to the endosomes and ligand-bound EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab5 and EEA1 (A), Rab5 and CD63 (B), and Rab5 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Right (A–C): single Z-plane of images. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab5 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab5 staining quantified in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Scale bars: 10 μm. p values were not statistically significant. Zoomed images correspond to the dashed inset boxes of the indicated images. Note: (B) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (B) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (B) and (C) is intentional.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition

doi: 10.1101/2020.08.18.255935

Figure Lengend Snippet: a , PLA between SARS-CoV-2 Spike and ACE2 in Calu-3 depleted of IFITM1, IFITM2 or IFITM3 and infected with genuine SARS-CoV-2. Lines represent means of n=2 (a) n=3 (b) (60-100 cells) ±SEM. b , PLA between Spike and ACE2 in Calu-3 cells depleted of IFITM2 and infected with SARS-CoV-2 virus on ice for 2 h and then incubated for 15 min at 37°C. Lines represent means of n=3 (200-300 cells) ±SEM. c , PLA assay between Spike and RAB5A in Calu-3 cells infected as in c . Lines represent means of n=2 (130-200 cells) ±SEM. DAPI (blue), nuclei. PLA signal (yellow). Scale bar, 20 µm. d , Quantification of ACE2-Spike and Spike-RAB5 alpha proximity upon SARS-CoV-2 infection.

Article Snippet: For staining following antibodies were used: IFITM1 (α-IFITM1 Cell Signaling 13126 S), IFITM2 (α-IFITM2 Abcam 236735), IFITM3 (α-IFITM3 Cell Signaling 59212S), SARS Spike CoV-2 (SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9], GTX-GTX632604), Rab5 alpha (Rab5 (RAB5A) Goat Polyclonal Antibody Origene AB0009-200) and ACE2 (Rabbit polyclonal anti-ACE2 Abcam, ab166755).

Techniques: Infection, Virus, Incubation

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 1. Characterization of SUN2-AS1. (A) A schematic representation of human SUN2-AS1 transcribed from the antisense strand of the protein-coding gene SUN2 on chromosome 22. (B) 5′- and 3′ RACE was conducted to determine the full length of SUN2-AS1. Full-length SUN2-AS1 was chemically synthesized and cloned into the pcDNA3.1(+) vector. Total RNA from ZIKV-infected A549 cells was extracted at 24 h.p.i. The RNA was then reverse transcribed and amplified using the SMARTer RACE cDNA amplification kit. (C) The CPAT (Coding Potential Assessment Tool) was used to predict the coding probability of SUN2-AS1. (D) 293T cells were transfected with pcDNA3.1-GFP-HA (vector control), SUN2-HA (positive control), pcDNA3.1-HA-SUN2-AS1, or pcDNA3.1-SUN2-AS1-HA plasmid. At 36 h post- transfection, cells were harvested for a Western blot analysis to detect anti-HA bands. Blots were representative of at three independent experiments. (E) The relative expression of human β-actin (cytoplasmic control), U6 (nuclear control), and the expression of SUN2-AS1 were analyzed by using qRT-PCR in the nuclear and cytoplasmic fractions.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Synthesized, Clone Assay, Plasmid Preparation, Infection, Reverse Transcription, Amplification, Transfection, Control, Positive Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 2. Differential expression of SUN2-AS1 induced by various viruses. (A) A549 cells were infected with ZIKV at MOI 3, and total RNAs were harvested at indicated time points (0, 6, 12, 18, and 24 h.p.i.) to detect the SUN2-AS1 level by qRT-PCR (n = 5). (B) A549 cells were infected with ZIKV at indicated MOI (0, 0.05, 0.5, 5, and 10), and total RNAs were harvested at 24 h.p.i. to detect the SUN2-AS1 level by qRT-PCR (n = 5). (C) A549 cells were infected with ZIKV, DENV2 NGC, JEV, VSV, or HSV-1 (MOI 3), and total RNAs were harvested at 24 h.p.i. to detect the SUN2-AS1 level by qRT-PCR (n = 5). (D) A549, Huh7, LN229, hMDM (monocyte-differentiated macrophages), or 293T cells were infected with ZIKV at MOI 3 and harvested for total RNA extraction at 24 h.p.i. qRT-PCR was performed to detect the level of SUN2-AS1. Human U6 level was measured as an internal control and normalized to uninfected cells (MOI 0, 0 h, or wt A549) (n = 3). Bio logically independent experiments were conducted. Data were shown as means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Quantitative Proteomics, Infection, Quantitative RT-PCR, RNA Extraction, Control

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 3. SUN2-AS1 is an inducible host lncRNA through the type I IFN pathway. (A) 293T cells were transfected with varying amounts of cellular RNA (without ZIKV infection) or viral RNA cocktail (from 0 to 1 μg), which was extracted by the QIAamp Viral RNA Mini Kit. The levels of SUN2-AS1 were determined by qRT-PCR at 24 h post-transfection (n = 5). (B) 293T cells were transfected with various plasmids encoding Zika virus nonstructural proteins (1.0 μg). The levels of SUN2-AS1 were determined by qRT-PCR at 24 h post-transfection. (C) A549 cells were treated with 400 ng/ml poly(I:C), and the total cells were collected at indicated time points for RNA extraction to detect the level of SUN2-AS1 using qRT-PCR. (D) A549 cells were treated with 500 units/mL of IFN-β for 24 h and total RNAs were harvested to determine the level of SUN2-AS1 by qRT-PCR. (E) Putative transcription factor binding sites on the promoter region of SUN2-AS1. (F-G) RNA Immunoprecipitation (RIP) was performed on A549 cells using NF-κB and STAT1 antibodies. qRT-PCR analysis for SUN2-AS1, NKILA (binding to NF-κB p65) (F), and LUCAT1 (binding to STAT1) (G) expression in A549 (n = 3). (H–L) Control cells, IFNAR1KO or STAT1KO cells were treated with 500 units/mL of IFN-β for 24 h. Cell lysates were collected for Western blot to detect the phospho-STAT1, STAT1 and MX1 protein level (H for IFNAR1KO cells, and I for STAT1KO cells). Total RNAs were harvested to determine the level of IFNB1, ISG15 and MX1 by qRT-PCR (J-L). (M) A549 control cells, IFNAR1KO cells, or STAT1KO cells were infected with mock or ZIKV (MOI 3). Total RNAs were harvested at 24 h.p.i. to detect the level of SUN2-AS1 by qRT-PCR. Human U6 level was measured as an internal control and normalized to uninfected cells. Biologically independent experiments were conducted. Data were shown as means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Transfection, Infection, Quantitative RT-PCR, Virus, RNA Extraction, Binding Assay, RNA Immunoprecipitation, Expressing, Control, Western Blot

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 4. SUN2-AS1 promotes ZIKV replication. (A) Two SUN2-AS1 knockout A549 cells were generated by CRISPR/Cas9 technology. The knockout effects of SUN2- AS1 were determined by qRT-PCR under both mock and ZIKV infection. (B) Cell viability of control, SUN2-AS1KO-1, and SUN2-AS1KO-2 cells was determined by CCK8 with or without ZIKV infection at 24 h. (C-E) Control and SUN2-AS1KO cells were infected with ZIKV at an MOI of 3 for 24 h. The viral RNA was extracted by QIAamp Viral RNA Mini Kit to detect the ZIKV RNA level by qRT-PCR (C). The cells and supernatants were harvested for Western blot (D) and plaque assay (E). (F) The pcDNA3.1 plasmid (empty vector) or pcDNA3.1-HA-SUN2-AS1 plasmid (1.0 μg) (SUN2-AS1OE) was transfected into A549 cells followed by infection with mock or ZIKV (MOI 3) for 24 h. Cells were harvested at 24 h.p.i., and the expression levels of SUN2-AS1 were determined by qRT-PCR. (G) Cell viability of pcDNA3.1 and SUN2-AS1OE cells was determined by CCK8 with or without ZIKV infection at 24 h. (H-J) pcDNA3.1 and SUN2-AS1OE cells were infected with ZIKV at MOI 3 for 24 h. The viral RNA level (H), E protein level (I), and titers of ZIKV particles (J) were measured by qRT-PCR, Western blot, and plaque assay, respectively. Human β-actin level was measured as an internal control and normalized to uninfected cells. All experiments were independently repeated three times. Data were shown as means ± S.D. *P < 0.1; ***P < 0.001; NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Knock-Out, Generated, CRISPR, Quantitative RT-PCR, Infection, Control, Western Blot, Plaque Assay, Plasmid Preparation, Transfection, Expressing

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 5. SUN2-AS1 is indispensable for flavivirus infection. (A-B) Control and SUN2-AS1KO cells were infected with DENV2 NGC (A) or JEV (B) (MOI = 5). The supernatant was collected at 24 h.p.i. The viral titers were determined using the focus-forming assay (FFA). (C-D) Vector control (pcDNA3.1) and SUN2-AS1OE cells were infected with DENV2 NGC (C) or JEV (D) (MOI = 5). The supernatant was collected at 24 h.p.i. and the viral titers were determined by focus forming assay (FFA). (E-F) Control and SUN2-AS1KO cells were infected with VSV (MOI = 1) or HSV-1 (MOI = 1). The supernatant of VSV (E) and HSV-1 (F) was collected at 24 h.p. i. and the viral titers were determined by the plaque-forming assay. All experiments were independently repeated three times. The data were shown as means ± S.D. *p ≤0.1; **p ≤0.01; NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Infection, Control, Focus Forming Assay, Plasmid Preparation

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 6. SUN2-AS1 inhibits the expression of ISGs via regulating the transcription of ISGs mRNA. (A) Control and SUN2-AS1KO cells were infected with ZIKV at an MOI of 3 for 24 h. Total cellular RNA was extracted by TRIzol to detect the mRNA level of IFNβ (A), ISG15 (B), MX1 (C), PKR (D), or OASL (E) by qRT-PCR. Human β-actin level was measured as an internal control and normalized to uninfected cells. (F-J) mRNA stability assay. Control and SUN2-AS1KO cells were treated with actinomycin D for 0, 3, 6, and 9 h. Cellular RNAs were extracted for qRT-PCR to measure the mRNA levels of GAPDH (F), ISG15 (G), MX1 (H), PKR (I), or OASL (J). Human β-actin level was measured as an internal control and normalized to untreated cells. Biologically independent experiments (n = 3) were conducted, and all data are shown as means ± S.D. P-values were calculated by one-way ANOVA. **P < 0.01, ***P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Stability Assay

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 7. The proviral role of SUN2-AS1 is mediated by suppressing ISGs. (A) Cytotoxic effect of DMSO and Ruxolitinib in control or SUN2-AS1KO cells was determined by CCK8. (B-E) Control and SUN2-AS1KO cells were infected with ZIKV at an MOI of 3 for 1 h. Then treated with 1 μM ruxolitinib. Cellular RNAs were extracted for qRT-PCR to measure the mRNA levels of ISG15 (B), MX1 (C), PKR (D), or OASL (E). Human β-actin level was measured as an internal control and normalized to uninfected cells. (F-H) Control and SUN2-AS1KO cells were infected with ZIKV (MOI = 3). Then treated with 1 μM ruxolitinib for 24 h. ZIKV RNA was extracted by the QIAamp Viral RNA Mini Kit and determine by qRT-PCR (F). Cell lysates were collected for Western blot to detect the MX1, PKR and ZIKV E protein level (G). And the supernatants were collected at 24 h.p.i for plaque assay (H). Biologically independent experiments (n = 3) were conducted, and all data are shown as means ± S.D. P-values were calculated by one-way ANOVA. *P < 0.1, **P < 0.01, ***P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Control, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 1. Effects of 3A5C7 monoclonal antibody (mAb) on morphine-induced mu-opioid receptor (MOR) endocytosis from cell membrane to cytoplasm. (A) Immunofluorescence staining indicated the endocytosis of MOR in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (B) Immunofluorescence staining indicated the endocytosis of MOR in SH- SY5Y cells treated with morphine and 3A5C7 mAb. (C) Immunofluorescence staining manifested the colocalization of MOR and Rab5 in HEK293T-MOR cells subjected to 3A5C7 mAb and morphine. (D, E) Flow cytometry showed the endocytosis of MOR in HEK293T-MOR cells co-treated with morphine and 3A5C7 mAb. (F, G) Flow cytometry showed the

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Membrane, Staining, Flow Cytometry

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 6. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance and physical dependence in mice. (A) Western blots indicating the cross specificity of 3A5C7 mAb against mu-opioid receptor (MOR) in the cortex, cerebellum, and hippocampus from C57/B6 mice. (B) Experiment flowchart for testing the effects of chronically administered 3A5C7 mAb on morphine tolerance and dependence. (C) The effects of chronically administered 3A5C7 mAb on the antinociceptive effects of morphine in mice measured by hotplate test. (D) The body weight changes of morphine-tolerant mice. Two-way analysis of variance with Bonferroni's post hoc tests were used for statistical analysis (n ¼ 6e7 mice in each group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, morphine þ mAb group vs. morphine group. #P < 0.05, ##P < 0.01, and ###P < 0.001, morphine þ mAb group vs. morphine þ normal IgG (NIg) group. (E) Representative immunofluorescence staining of MOR and Rab5 in the mouse dorsal root ganglions (DRGs) after

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Western Blot, Staining

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 7. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance via G protein-coupled receptor kinase 2 (GRK2)/b-arrestin2 pathway in mice. (A) Immunoblot showing that GRK2 was knocked-down by short hairpin ribonucleic acid (shRNA) in dorsal root ganglions (DRGs). (B) Quantification of the protein level of GRK2 in Fig. 7A. (C) Immunoblot showing that b-arrestin2 was knocked-down by shRNA in DRGs. (D) Quantification of the protein level of b-arrestin2 in Fig. 7C. (E) Hotplate tests demonstrating the effects of GRK2 or b-arrestin2 knockdown on the anti-tolerance efficacy of mAb 3A5C7. Two-way analysis of variance (ANOVA) with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ****P < 0.0001, sh-NC þ morphine þ mAb group vs. sh-NC þ morphine group; ###P < 0.001 and ####P < 0.0001, sh- NC þ morphine þ mAb group vs. sh-GRK2 þ morphine þ mAb group; &P < 0.05 and &&&P < 0.001, sh-NC þ morphine þ mAb group vs. sh-b-arrestin2 þ morphine þ mAb group (n ¼ 5 mice in each group). (F) Representative immunofluorescence images of MOR and Rab5 for each treatment in mouse DRGs (n ¼ 3 mice in each group). (G) The protein levels of hippocampal protein kinase A (PKA) from mice in each treatment group. (H, I) Quantification of the relative protein levels of PKA from Fig. 7G. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ***P < 0.001. (J) Inhibitory effects of acute 3A5C7 mAb administration on naloxone-precipitated withdrawal jumping were reduced in GRK2- and b-arrestin2-knockdown mice. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. ****P < 0.0001 vs. sh-NC þ morphine group; ####P < 0.0001 vs. sh-NC þ morphine þ mAb group (n ¼ 5 mice in each group). Data were presented as the mean ± standard error of mean. sh-NC: control shRNA; sh-GRK2: shRNA for GRK2; sh-b-arrestin2: shRNA for b-arrestin2; MPE: maximum possible effect; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Western Blot, shRNA, Knockdown, Control