Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Sequence alignment of the A333-N361 region using Clustal Omega to all available Cx45 RefSeq sequences revealed a high degree of conservation within this region, with 72.4% identical, 20.6% conserved, and 6.9% non-conserved residues. For clarity, the sites of point mutations in the Cx45 6E construct are indicated by the single letter code above the alignments. Degree of site conservation (excluding Cx45 6E mutations) is indicated at the bottom, * = identical, : and . = high conservation and intermediate conservation, respectively.

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Sequencing, Construct

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Three different MDCK cells expressing either Cx45 WT or 6E were assessed for expression at the protein and transcript levels. A) Western blots and quantification of Cx45 WT, Cx45 6E, and Cx43 expression. B) RT-PCR gel images and quantification of Cx45 WT and 6E transcripts. C) Western blots and quantification of Cx45 WT or 6E subjected to a 12 hr cycloheximide chase. Results presented as the mean + s.e.m. (n=3). Statistics are used to analyze the data were two-tailed unpaired Student’s T-test (A, n=3, *P<0.05; and C, n=3, n.s., *P<0.9056) and one-way ANOVA with a Neuman-Keuls post hoc test (B, n=3; *P<0.05, **P<0.001 comparing Cx45 WT and 6E, and ##P<0.001 compared to time = 0).

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Representative immunofluorescence images (red) of methanol fixed cells stably expressing Cx45 WT (left) or 6E (right). Co-immunostaining (green) for either E-Cadherin (MDCK) or β-catenin (Hek-293T) was used to mark the plasma membranes of the fixed cells (blue = DAPI). A) Clonally selected MDCK cells stably expressing mouse Cx45 WT or 6E. B) Bulk selected MDCK cells stably expressing human Cx45 WT or 6E. C) Bulk selected Hek-293T cells stably expressing mouse Cx45 WT and 6E. D) Isolated time-lapse frames of clonally selected MDCK cells expressing C-terminal eGFP fusions of mouse Cx45 WT or 6E (green). White arrows in the insets indicate gap junction plaques. Scale bar A–C = 20 μm and D = 10 μm.

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Immunostaining, Isolation

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Triton X-100 solubility was used to compare MDCK cells expressing Cx45 WT or 6E. A) Western blot image of Triton X-100 extracted protein; total lysate (T), soluble (S), and insoluble (I) fractions. Below is the quantification of protein in the T, S, and I fractions. Statistics represent one-way ANOVA with a Neuman-Keuls post-hoc correction (n=3, **P<0.001,***P<0.0001). B) Representative fluorescent images (left) and quantification (right) of in situ Triton X-100 and mock (1x PBS) extracted Cx45 WT or 6E in MDCK cells (red, Cx45; blue, DAPI). C) MDCK cells expressing Cx45 WT (top) or 6E (bottom) were scrape loaded with tracer dyes to assess the degree of intercellular communication. Red, Texas red dextran (MW 10,000 Da); yellow, Lucifer yellow CH (MW 443 Da; −2 charge), green, neurobiotin (MW 287 Da; +1 charge); and blue, DAPI. Images are representative of greater than six independent experiments. Scale bar in B = 20 μm and C = 100 μm.

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Solubility, Expressing, Western Blot, In Situ

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: MDCK cells stably expressing Cx45 WT or 6E were subjected to conditions that activate hemichannels activity (Ca2+ deprivation) and the level of neurobiotin uptake was measured. Representative fluorescent micrographs of cells loaded with neurobiotin and probed with streptavidin-647 conjugate (red). Quantification of DAPI (blue) normalized mean fluorescent intensity (MFI) from 3 replicates, 15 random 100X FOVs were acquired per replicate. Statistics are one-way ANOVA with a Neuman-Keuls multiple comparison test (***P<0.0001 relative to +Ca2+ within same group, ^ and ^^^P <0.05 and <0.0001 respectively, relative to −Ca2+ within same group; ###P<0.0001 between −Ca2+ treatment groups; •••P<0.0001 compared to WT + pervanadate). Scale bar = 50 μm.

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Stable Transfection, Expressing, Activity Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: MDCK cells stably expressing Cx45 WT or 6E were cultured, lysed, and immunoprecipitated to assess total phosphorylation levels and interactions with β-tubulin, Drebrin, and Nedd4. A) Representative Western blots of immunoprecipitated Cx45 WT or 6E blotting with a general anti-pY antibody. (n=3, ** P<0.01) Representative Western blot of B) β-tubulin (n=7, ***P<0.001), C) Drebrin (n=3, **P<0.01), and D) Nedd4 (n=3, **P<0.01) co-immunoprecipitated with either Cx45 WT or 6E. Data presented as mean fold change + s.e.m. relative to the first WT replicate. Statistics are two-tailed unpaired Student’s T-test.

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Stable Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Journal of molecular and cellular cardiology

Article Title: Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

doi: 10.1016/j.yjmcc.2017.07.010

Figure Lengend Snippet: Oocytes injected with equal amounts of cRNA coding for Cx45 WT or the 6E mutant were voltage clamped and hemichannel currents elicited by large depolarizing voltage steps from −50 to +80 mV in 10 mV incrememts from a holding potential of −80 mV. A) Representation of voltage protocol. Under the same voltage protocols, the WT channels mediated substantially less current than the 6E channels. Representative current plots from (B) WT and (C) 6E expressing oocytes.

Article Snippet: α-Cx45-CT(MAB3101), α-mouse-HRP (H+L; 12-439), α-pS (05-1000), and α-Nedd4 (07-049) were purchased from EMD Millipore; α-Cx43 (C6219), α-β-catenin (C2206), and α-β-tubulin (T5201) were purchased from Sigma-Aldrich; α-E-cadherin-Alexa488 (#3195), α-HA-647 (#3444), α-Myc-555 (#3756), α-rabbit-Alexa555 (#4413), α-mouse-Alexa647 (#4410), Normal Rabbit IgG (#2729), and α-pY-1000 Multi-Mab (#8954) were purchased from Cell Signaling; α-mouse-Alexa488 (A-11001), Texas Red Dextran (10,000 Da; D1863), Lucifer yellow CH (L453), and Streptavidin-Alexa647 ( {"type":"entrez-protein","attrs":{"text":"S21374","term_id":"99986","term_text":"pir||S21374"}} S21374 ) were purchased from Life Technologies; α-rabbit-HRP (H+L; #20320) was purchased from Alpha Diagnostics; Neurobiotin (SP-1120) was purchased from Vector Labs; α-rabbit-HRP (Light chain; #211-032-171) was purchased from Jackson Immuno Research; α-Cx45-CL (ACC-207) was purchased from Alomone; Veriblot-HRP (ab131366) was purchased from Abcam; and DAPI (#02157574) was purchased from MP Biomedicals.

Techniques: Injection, Mutagenesis, Expressing

Journal: Journal of Virology

Article Title: Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101

doi: 10.1128/jvi.00438-23

Figure Lengend Snippet: RNAi screening identifies TSG101 as an ESCRT factor supporting RABV infection. (A) Schematic image of the RNAi screening method. siRNA-transfected SK-N-SH cells were infected with RABV-Nluc at an MOI of 10, and culture supernatants collected at 18 hpi were passaged into NA cells. Luciferase activity in the NA cells was measured at 8 hpi. (B) Luciferase activity derived from NanoLuc-encoded reporter RABV in siRNA-treated SK-N-SH cells relative to the luciferase activity in control siRNA-treated cells. Dots indicate the mean of three different siRNAs for each target. Bars indicate the means ± standard deviations of the three siRNAs. (C) Virus titers in the supernatants of TSG-KD SK-N-SH cells at 48 hpi. siRNA-treated cells were infected with RABV at an MOI of 1. The titers were measured using a focus-forming assay. Bars indicate the means ± standard deviations of three replicates from a representative experiment. (D) Schematic images of the TSG101 mutants used in this study. Mutation sites are marked in red. UEV, ubiquitin-conjugating enzyme E2 variant; PRD, proline-rich domain; CC, coiled-coil domain; PTAP, conserved PTAP tetrapeptide motif; SB, steadiness box. (E) Virus titers in TSG-KD and rescue cells. TSG-KD cells were transfected with siRNA-resistant TSG101-encoding plasmids and infected with RABV at an MOI of 1. The virus titers in supernatants at 24 hpi were measured. Bars indicate the means ± standard deviations of three replicates from a representative experiment. For statistical analyses, Welch’s t test used in panel C (*, P < 0.05), and one-way ANOVA and Dunn’s multiple-comparison tests were used in panel E (*, P < 0.05; ***, P < 0.001).

Article Snippet: SK-N-SH cells were reverse transfected with 20 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) in collagen-coated 48-well plates and then cultured for 60 h. Knockdown efficacy was checked by immunoblotting (anti-Alix antibody [catalog no. 92880; Cell Signaling], anti-Nedd4 antibody [catalog no. 2740; Cell Signaling], anti-Nedd4-like antibody [catalog no. 4013; Cell Signaling], and anti-TSG101 antibody [catalog no. 28283-1-AP; Proteintech]) or qRT-PCR. siRNA-treated cells were infected with virus at an MOI of 1, and the supernatants were collected at 48 to 60 hpi.

Techniques: Infection, Transfection, Luciferase, Activity Assay, Derivative Assay, Control, Virus, Focus Forming Assay, Mutagenesis, Ubiquitin Proteomics, Variant Assay, Comparison

Journal: Journal of Virology

Article Title: Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101

doi: 10.1128/jvi.00438-23

Figure Lengend Snippet: Downregulation of TSG101 expression obstructs the RABV budding process. (A) Virus attachment on the surface of TSG-KD cells. SK-N-SH cells were incubated with RABV at 4°C for 1 h. After the cells were washed, RNA was extracted with attached virions and analyzed using qRT-PCR. (B) Viral entry into TSG-KD cells. SK-N-SH cells were infected with RABV at an MOI of 10. After incubation at 37°C for 30 min, uninternalized virions were removed via trypsin treatment. Internalized virions were measured using qRT-PCR. (C) RABV minigenome replication in TSG-KD cells. 293T cells exogenically expressing the RABV minigenome were transfected with siRNA against TSG101. Minigenome replication was evaluated by measuring the luminescence signal from NanoLuc. (D) Viral RNA levels at the early stage of virus infection. Viral RNA levels in TSG-KD SK-N-SH cells were measured at the indicated time points using qRT-PCR. (E) Virus titers at the early stage of virus infection. Virus titers in the supernatants from TSG-KD SK-N-SH cells were measured at the indicated time points. (F) Focus size of RABV-infected TSG-KD A549 cells. Foci formed by RABV-infected cells were immunostained with anti-RABV N antibody at 72 hpi. Scale bar, 200 μm. The areas of 30 foci selected randomly were measured using ImageJ. (G) Number of foci in TSG-KD A549 cells. (H) Localization of RABV M in TSG-KD SK-N-SH cells. Cells infected with RABV were immunostained with anti-RABV M antibody at 24 hpi and analyzed using confocal microscopy. Scale bar, 20 μm. (I) Electron microscopic images of RABV-infected TSG-KD SK-N-SH cells at 28 hpi. Arrowheads, virions; dotted line, accumulation of virions. Scale bar, 500 nm. (J) Purified RABV virions were negatively stained and analyzed using electron microscopy. Scale bar, 200 nm. (K) Virion diameter and abundance ratio. Purified RABV virions in 50 images captured randomly with an electron microscope were measured using ImageJ. (A to G) Means ± standard deviations of three replicates from a representative experiment. Statistical analyses in panels A, B, F, and G were performed by Welch’s t test (*, P < 0.05; ****, P < 0.0001); those in panel C were performed by one-way ANOVA and Dunnett’s multiple-comparison test (*, P < 0.05); and those in panels D and E were done by multiple t -tests (*, P < 0.05). ns, not significant.

Article Snippet: SK-N-SH cells were reverse transfected with 20 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) in collagen-coated 48-well plates and then cultured for 60 h. Knockdown efficacy was checked by immunoblotting (anti-Alix antibody [catalog no. 92880; Cell Signaling], anti-Nedd4 antibody [catalog no. 2740; Cell Signaling], anti-Nedd4-like antibody [catalog no. 4013; Cell Signaling], and anti-TSG101 antibody [catalog no. 28283-1-AP; Proteintech]) or qRT-PCR. siRNA-treated cells were infected with virus at an MOI of 1, and the supernatants were collected at 48 to 60 hpi.

Techniques: Expressing, Virus, Incubation, Quantitative RT-PCR, Infection, Transfection, Confocal Microscopy, Purification, Staining, Electron Microscopy, Microscopy, Comparison

Journal: Journal of Virology

Article Title: Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101

doi: 10.1128/jvi.00438-23

Figure Lengend Snippet: RABV M interacts with TSG101 via the L-domain. (A) Schematic representation of RABV M and the L-domain mutants. (B) Colocalization of RABV M and TSG101. SK-N-SH cells stably expressing TSG101-Venus were infected with RABV, immunostained with anti-RABV M at 24 hpi, and analyzed using confocal microscopy. Arrowheads show colocalization. Scale bar, 50 μm. (C to F) Coimmunoprecipitation of RABV M with TSG101. HA- or FLAG-tagged RABV M and TSG101 were coexpressed in 293T cells and coimmunoprecipitated using anti-HA magnetic beads. Immunoblotting was performed with anti-HA or -FLAG antibody. Bar graphs show the relative precipitation efficacy (IP/input) of FLAG compared with that of HA from a representative experiment following quantification via ImageJ.

Article Snippet: SK-N-SH cells were reverse transfected with 20 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) in collagen-coated 48-well plates and then cultured for 60 h. Knockdown efficacy was checked by immunoblotting (anti-Alix antibody [catalog no. 92880; Cell Signaling], anti-Nedd4 antibody [catalog no. 2740; Cell Signaling], anti-Nedd4-like antibody [catalog no. 4013; Cell Signaling], and anti-TSG101 antibody [catalog no. 28283-1-AP; Proteintech]) or qRT-PCR. siRNA-treated cells were infected with virus at an MOI of 1, and the supernatants were collected at 48 to 60 hpi.

Techniques: Stable Transfection, Expressing, Infection, Confocal Microscopy, Magnetic Beads, Western Blot

Journal: Journal of Virology

Article Title: Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101

doi: 10.1128/jvi.00438-23

Figure Lengend Snippet: The RABV YL motif is essential for TSG101-mediated viral growth. (A) Schematic representation of recombinant RABV with alanine substitutions in the L-domain in RABV M. (B) Virus titers of RABV L-domain mutants. TSG-KD SK-N-SH cells were infected with RABV mutants at an MOI of 1, and virus titers in the supernatants at 48 hpi were measured. (C) Fold change of virus titers of RABV mutants compared to that of wild-type virus. Means ± standard deviations of three replicates from a representative experiment. Ordinary one-way ANOVA with Dunnett’s multiple-comparison test; ***, P < 0.001. (D) Immunoblotting of TSG101 in purified virions. (E) Colocalization of RABV M and TSG101. SK-N-SH cells stably expressing TSG101-Venus were infected with RABV, immunostained with anti-RABV M at 24 hpi, and analyzed using confocal microscopy. Arrowheads show colocalization. Scale bar, 50 μm.

Article Snippet: SK-N-SH cells were reverse transfected with 20 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) in collagen-coated 48-well plates and then cultured for 60 h. Knockdown efficacy was checked by immunoblotting (anti-Alix antibody [catalog no. 92880; Cell Signaling], anti-Nedd4 antibody [catalog no. 2740; Cell Signaling], anti-Nedd4-like antibody [catalog no. 4013; Cell Signaling], and anti-TSG101 antibody [catalog no. 28283-1-AP; Proteintech]) or qRT-PCR. siRNA-treated cells were infected with virus at an MOI of 1, and the supernatants were collected at 48 to 60 hpi.

Techniques: Recombinant, Virus, Infection, Comparison, Western Blot, Purification, Stable Transfection, Expressing, Confocal Microscopy

Journal: Cellular and molecular life sciences : CMLS

Article Title: IGF2BP1-HAX-1 positive feedback loop-mediated HAX-1 overexpression blocks autophagic flux and promotes chemoresistance in nasopharyngeal carcinoma.

doi: 10.1007/s00018-025-05604-0

Figure Lengend Snippet: Figure 8 Schematic overview of the role of HAX-1 in DDP chemoresistance by effectively blocking autophagic flux in NPC. IGF2BP1 enhanced HAX-1 m6A methylation, thereby enhancing its stability. Furthermore, HAX-1 binds IGF2BP1 mRNA, thereby contributing to its stability and translation. Moreover, HAX-1 recruits NEDD4 to promote Rab7a degradation and inhibits binding of Rab7a with SNAREs by competitively binding to it, thereby blocking the fusion of autophagosomes with lys- osomes

Article Snippet: Co-IP co-immunoprecipitation, WB western blot ◂ Reagent or Resource Source Identifier IGF2BP1 proteintech 22803-1-AP NEDD4 proteintech 21698-1-AP HA-Tag Cell Signaling Tech- nology #3724 Myc-Tag Cell Signaling Technology #2276 YTHDF1 proteintech 17479-1-AP YTHDF2 proteintech 24744-1-AP WTAP proteintech 60188-1-Ig GAPDH proteintech 10494-1-AP Bacterial and Virus Strains lentiviral HAX1- shRNA Shanghai Genechem Target seq: GAG TGA TGC AAG AAG TGA A lentiviral IGF2BP1shRNA Shanghai Genechem Target seq: ACA GTA GAG AAC TGT GAG CAA lentiviral Rab7AshRNA Shanghai Genechem Target seq: gaAAC AAG ATT GAC CTC GAA A RFP-GFP-tagged LC3 Shanghai Genechem tfLC3 Biological Samples Mouse tissues This paper N/A Human NPC speci- mens Affiliated Hospital of Nantong University N/A Chemicals, peptides, and recombinant proteins DMSO Fisher Scientific Cat#BP231-100 Cisplatin Jiangsu HANSOH PHARMA H20040813 Hochest Thermo Fisher Scientific 62249 Propidium Iodide Becton Dickinson and Company 556463 Fetal Bovine Serum Biological Industries 04-001-1ACS Reagent or Resource Source Identifier RPMI 1640 Biological Industries 01–100-1ACS TRIzol reagent BBI B511311 Uranyl acetate Polysciences, Hirschberg an der Bergstrasse, Germany 6159-44-0 Basic lead citrate Sigma-Aldrich, Taufkirchen, Germany 15326 Actinomycin D MCE, Sollentuna, Sweden HY-17559 Cycloheximide MCE, Sollentuna, Sweden HY-12320 MG-132 MEC, Sollentuna, Sweden HY-13259 AR6 buffer AKOYA, Marlborough, Massachusetts, USA AR600 CQ, chloroquine MCE HY-17589A Critical commercial assays EdU cell prolifera- tion assay Shanghai Beyotime C0078S CCK8 kit BBI E606335 LIVE/DEAD viabil- ity kit Shanghai Bestbio BB-4126 BCA protein assay kit Thermo Fisher Scientific 23227 Annexin V-FITC Apoptosis Detection Kit Beyotime C1063 Dual-Luciferase Reporter Assay Kit Beyotime RG088M Oligonucleotides Primer1 for HAX1, Forward CAG GAG GAG GGA TAC GTT TCC This paper N/A Primer1 for HAX1, Reverse CCC ATA TCG CTG AAG ATG CTATT This paper N/A Primer1 for preHAX1, Forward ATT ACA GTC GCC TGC CAA AC This paper N/A Primer1 for preHAX1, Reverse AGG TCA GGA GCT CAA AAG CA This paper N/A Primer1 for IGF2BP1, Forward GCG GCC AGT TCT TGG TCA A This paper N/A Primer1 for IGF2BP1, Reverse TTG GGC ACC GAA TGT TCA ATC This paper N/A Figure 5 Regulation of IGF2BP1 mRNA stability and translation by IGF2BP1.

Techniques: Blocking Assay, Methylation, Binding Assay