Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: Human retina tissue expression and iPS-RPE cell expression patterns of CL-11. ( a ) Representative confocal image showing CL-11 expression on permeabilised (left panel) and non-permeabilised (middle panel) human retinal sections. The sections shown are taken from the peripheral retina and from the central retina outside the macula. Control sections were incubated with no primary antibody (right panel). CL-11 (green), nuclei (blue) and autofluorescence (red) are shown. Scale bars, 25 μm. CL-11 staining without DAPI is shown on the right side of peripheral retina and central retina panels. High magnification images of area indicated in the white box are shown in the bottom panel. Scale bar, 10 μm. ( b ) Representative confocal microscopic image showing differentiated iPS-RPE cells morphology (left panel) and CL-11 staining on permeabilised human iPS-RPE cells (middle and right panel). CL-11 (red), nuclei (blue) and phalloidin (aqua) are shown. Scale bars, 25 μm ( c ) Flow cytometry histograms showing CL-11 expression on permeabilised iPS-RPE cells (intracellular) versus non-permeabilised cells (surface). Cells stained with secondary antibody alone were used as control.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Expressing, Incubation, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: CL-11 binding to hypoxia-stressed iPS-RPE cells. ( a ) Representative Western blot under reducing conditions showing CL-11 expression in iPS-RPE cells under normal and hypoxic conditions. HSP90 was used as a loading control. Full-length blots are presented in Supplemental Fig. . ( b ) Relative density analysis of CL-11 in iPS-RPE cells following different treatments (N = 3 independent experiments). ( c ) Detection of both non-reduced (100 kDa) and reduced states (34 kDa) of CL-11 in the culture supernatants of iPS-RPE cells following or not hypoxic stress by Western blotting. ( d ) Representative immunofluorescence images showing CL-11 binding to the surface of hypoxic-stressed iPS- RPE cells compared to non-stressed cells. Scale bars, 25 μm. ( e ) Quantification of CL-11 binding in normal and hypoxic iPS-RPE cells using ImageJ software. CL-11 (red), nuclei (blue) and phalloidin (aqua) are shown. N = 3 independent experiments, * **P < 0,0001, t -test. ( f ) Confocal images of CL-11 (red) and L-fucose (green) showing co-expression of CL-11 and L-fucose (orange) on hypoxic–iPS-RPE cells treated or untreated with fucosidase. Scale bars, 50 μm. ( g ) Quantification of L-fucose expression and ( h ) CL-11 binding to hypoxia-stressed cells treated or untreated with fucosidase using ImageJ software. N = 4 independent experiments.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Binding Assay, Western Blot, Expressing, Immunofluorescence, Software

Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: C3d deposition and Mac assembly on hypoxia-stressed iPS-RPE cells. ( a ) Representative Western blot showing C3 protein expression in iPS-RPE cells under normal and hypoxic conditions. Actin was used as a loading control. Full-length blots are presented in Supplemental Fig. . ( b ) Representative immunofluorescence images showing CL-11 (red) and C3d (green) staining on hypoxia-stressed iPS- RPE cells compared to non-stressed cells. Nuclei (blue) and phalloidin (grey) are shown. Scale bars, 25 μm. Quantification of CL-11 ( c ) and C3d ( d ) expression following or not hypoxia stress using ImageJ software. N = 3 independent experiments; n = 15 images analysed * **P < 0,0001, t -test. ( e ) CL-11 and C3d co-localization was quantified by calculating Mander’s co-localization coefficients using JACoP plug-in and ImageJ software. ( f ) Representative confocal images of CL-11 (green), MAC (red) nuclei (blue) and phalloidin (aqua) showing co-expression of CL-11 and MAC on hypoxic iPS-RPE compared to non-stressed cells. Scale bars, 25 μm. High magnification image (white box) showing co-localization of CL-11 and MAC. Scale bars, 10 μm. ( g ) CL-11 and MAC co-localization was quantified by calculating Mander’s co-localization coefficients using JACoP plug-in and ImageJ software. N = 3 independent experiments.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Software

Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: CL-11 and MASP-2 interaction on hypoxia-stressed iPS-RPE cells. ( a ) Representative Western blot showing MASP-2 protein expression in iPS-RPE cells under normal and hypoxic conditions. HSP90 was used as a loading control. Full-length blots are presented in Supplemental Fig. . ( b ) Relative density analysis of MASP-2 in iPS-RPE cells under different treatments. N = 3 independent experiments. ( c ) Representative immunofluorescence images showing MASP-2 staining on hypoxia-stressed iPS- RPE cells compared to non-stressed cells. MASP-2 (red) and phalloidin (aqua) are shown on the left panels and MASP-2 (red) alone is shown on the right panels. Scale bars, 25 μm. ( d ) Representative immunofluorescence images showing co-expression of CL-11 and MASP-2 (orange) on the surface of hypoxia-stressed iPS- RPE cells MASP-2 (red), CL-11 (green) nuclei (blue) and phalloidin (grey) are shown. Scale bars, 25 μm. ( e ) CL-11 and MASP-2 co-localization was quantified by calculating Mander’s co-localization coefficients using JACoP plug-in and ImageJ software.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Software

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Imaging of Human Insulin Secreting Cells with Gd-DOTA-P88, a Paramagnetic Contrast Agent Targeting the Beta Cell Biomarker FXYD2γa

doi: 10.3390/molecules23092100

Figure Lengend Snippet: Expression of FXYD2γa in pancreatic human islets and EndoC-βH1 cells. ( A ) Quantitative RT-PCR (qPCR) of FXYD2γa mRNA expression in EndoC-βH1 cells ( n = 5) and human pancreatic islets ( n = 4). Data are presented as a box-plot. ( B ) A representative immunoblot of EndoC-βH1 cells, with alpha-tubulin as a reference protein ( n = 3); ( C ) Immunocytochemistry of EndoC-βH1 cells showing surface localization of FXYD2γa (green) and Hoechst staining of nuclei (blue) ( n = 3). The negative staining control of EndoC-βH1 cells (without the FXYD2γa antibody) is presented at the right side of panel C; the white scale bar represents 1 µm. The luminosity and the contrast were uniformly increased in both pictures to improve visualization. The original pictures are shown in ( D – I ). Histological evaluation of the implanted EndoC-βH1 tumors, showing cells clustered into pseudo-islets surrounded by fibrotic tissue ( D ), capillary networks (yellow arrows) ( E ), insulin expression ( F ) with its corresponding negative control ( G ) and FXYD2γa detected with either SPY393 polyclonal antibody ( H ) or the biotinylated P88 ( I ); ( D – I ) are representative micrographs from 2 EndoC-βH1 cell-implanted mice.

Article Snippet: For FXYD2γa detection, addition of the rabbit pAb SPY393 (12.5 µg/mL; Eurogentec) was followed by fluorescein goat anti-rabbit IgG (20 μg/mL; Vector Labconsult, Brussels, Belgium).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Staining, Negative Staining, Negative Control

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: SERPINB1 promotes SVA replication via a specific functional region. Overexpression of SERPINB1 promotes SVA replication. IBRS-2 cells were transfected with SERPINB1 expression plasmid pSERPINB1-Myc and/or vector, and infected with 0.1 MOI SVA at 24 hpt. After incubation for 24 h, the expression of viral VP2 protein and SERPINB1 protein was determined by western blot with anti-VP2 and anti-Myc (A). Viral RNA (B) and virus yields (C) were assessed by qRT-PCR and TCID50, respectively. Knockdown of SERRPINB1 inhibits SVA replication. IBRS-2 cells were transfected with siRNA against SERPINB1, and cells were inoculated with 0.1 MOI SVA for 24 h. SVA VP2 and endogenous SERPINB1 were assayed using a western blot. The efficiency of knockdown, siSERPINB1−3 > siSERPINB1-2 > siSERPINB1-1 (D). Viral RNA (E) and virus yields (F) were assessed by qRT-PCR and TCID50, respectively. (G) Diagram showing SERPINB1 domains and gene truncation constructs. (H and I) Transfected IBRS-2 cells were challenged with SVA (0.1 MOI) for 24 h, and then protein extracts were analyzed using western blotting. ImageJ was used to quantify the level of protein, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (J) Diagram showing SERPINB1 mutant sequence. (K) Transfected IBRS-2 cells were challenged with SVA (0.1 MOI) for 24 h, and then protein extracts were analyzed using western blotting. ImageJ was used to quantify the level of protein, and GAPDH was used as a loading control. (L) Virus yields presented as TCID50 per milliliter. All samples run in triplicate. ns, not significant; *, P < 0.05; **, P < 0.01, ***, P < 0.001.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Functional Assay, Over Expression, Transfection, Expressing, Plasmid Preparation, Infection, Incubation, Western Blot, Virus, Quantitative RT-PCR, Knockdown, Construct, Control, Mutagenesis, Sequencing

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: SERPINB1 negatively regulates the type I IFN signaling pathway and positively regulates autophagy. (A–C) SERPINB1 attenuates the type I IFN signaling pathway. pSERPINB1-Myc and/or empty vector-transfected IBRS-2 cells with or without stimulus of SeV, Poly(I:C), and SVA. The cells were harvested for RNA extraction and qRT-PCR detection of IFN-β mRNA levels. (D) SERPINB1 induces autophagy. Western blot displaying proteins from pSERPINB1-Myc and/or empty vector-transfected IBRS-2 cells. Intracellular autophagy levels were probed with antibodies against AKT, p-AKT, mTOR, p-mTOR, and LC3. ImageJ was used to quantify the level of protein, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each sample was run in triplicate. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Plasmid Preparation, Transfection, RNA Extraction, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: SVA replication, type I IFN, and autophagy are changed in SERPINB1-deficient HEK-293T cells. (A) SERPINB1 inhibits the type I IFN signaling pathway in HEK-293T cells. Measurements were obtained using pSERPINB1-Myc or empty vector-transfected HEK-293T cells with or without the SeV infection. The cells were harvested for RNA extraction and qRT-PCR analysis of IFN-β, ISG15, and ISG56 mRNA levels. (B) Diagram showing construction of SERPINB1-deficient HEK-293T cells. (C) Western blot analysis of SERPINB1 expression in SERPINB1 KO and control cells. (D) SVA replication was downregulated in SERPINB1-deficient HEK-293T cells. SERPINB1+/+ and SERPINB1−/− HEK-293T cells were transfected with pSERPINB1-Myc or empty vector, then infected with SVA (0.1 MOI) for 24 h. The cells were harvested for western blot analysis. (E–G) The IFN pathway response was enhanced in SERPINB1−/− HEK-293T cells. SERPINB1+/+ and SERPINB1−/− HEK-293T cells were analyzed with or without stimulus. The cells were harvested for RNA extraction and qRT-PCR analysis of IFN-β mRNA levels. (H) Western blot analysis of intracellular autophagy levels of SERPINB1+/+ and SERPINB1−/− HEK-293T cells. ImageJ was used to quantify the level of protein, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The data presented are means ± SEM from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01, ***, P < 0.001.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Plasmid Preparation, Transfection, Infection, RNA Extraction, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: SERPINB1 binds IKBKE (A–C) SERPINB1 interacts with IKBKE in a specific way. (A) Immunoprecipitation and western blot of HEK-293T cells co-transfected with adaptor proteins and pSERPINB1-Myc. Proteins were immunoprecipitated with rabbit anti-Myc. (B) pIKBKE-Flag and pSERPINB1-Myc co-transfected cell lysates were collected, and then Co-IP was performed using rabbit anti-Myc and mouse anti-Flag. (C) HEK-293T cells transfected with pIKBKE-Flag and/or vector were lysed for Co-IP analysis. Endogenous SERPINB1 was measured using a western blot. (D) Diagram showing SERPINB1 domains and gene truncation constructs. (E) Cells expressing SERPINB1 truncations and IKBKE proteins were harvested for Co-IP and western blot, and then proteins were immunoprecipitated with rabbit anti-Flag.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Transfection, Co-Immunoprecipitation Assay, Plasmid Preparation, Construct, Expressing

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: SERPINB1 degrades IKBKE through the proteasome pathway (A and B) SERPINB1 degrades IKBKE. (A) HEK-293T cells were co-transfected with pIKBKE-Flag (200 ng) along with increasing doses of pSERPINB1-Myc (200, 400, or 800 ng). The cells were harvested for western blot analysis. (B) HEK-293T cells overexpressed SERPINB1 and were subsequently treated with SeV. Endogenous IKBKE was measured using a western blot. ImageJ was used to quantify the level of protein, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) SERPINB1 truncations and pIKBKE-Flag were co-transfected into HEK-293T cells, and then cells were collected for western blot by anti-Flag, anti-Myc, and anti-GAPDH. ImageJ was used to quantify the level of protein, and GAPDH was used as a loading control. (D) HEK-293T cells were transfected with pIKBKE-Flag together with empty vector or pSERPINB1-Myc. At 12 hpt, cells were treated with Dulbecco’s modified Eagle medium or 3MA for 12 h. The cells were harvested for western blot analysis. ImageJ was used to quantify the level of protein, and GAPDH was used as a loading control. (E) HEK-293T cells were transfected with pIKBKE-Flag together with an empty vector or pSERPINB1-Myc. At 12 hpt, cells were treated with dimethylsulfoxide or MG132 (10 μM) for 12 h. The cells were harvested for western blot analysis. ImageJ was used to quantify the level of protein, and GAPDH was used as a loading control. SERPINB1 ubiquitinates IKBKE. (F) HEK-293T cells, transfected with pIKBKE-Flag or pTBK1-Flag, together with pSERPINB1-Myc, and pC3-Ub-HA, were subjected to Co-IP with anti-Flag. Western blot analysis was performed with antibodies against HA, Flag, and Myc-tag. (G) HEK-293T cells, transfected with pIKBKE-Flag or pTBK1-Flag, together with pSERPINB1-Myc, and pC3-K48-HA, were subjected to Co-IP with anti-Flag. Western blot analysis was performed with antibodies against HA, Flag, and Myc-tag. Each sample was run in triplicate. ns, not significant; *, P < 0.05; **, P < 0.01. ***, P < 0.001.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Transfection, Western Blot, Control, Plasmid Preparation, Modification, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: SERPINB1 pro-viral activity disappears when autophagy is inhibited. (A) SERPINB1 inhibits IFN signal via BECN1-mediated autophagy. Dual-luciferase reporter assays were used to examine the effect of SERPINB1 on the activity of the IFN-β promoter in BECN1+/+ and BECN1−/− HEK-293T cells. HEK-293T cells (WT or KO) were co-transfected with pIFN-β-Luc and pGL-TK along with pSERPINB1-Myc or vector and stimulated with SeV. The cells were harvested for a luciferase assay. (B–C) SERPINB1 promotes SVA replication that depends on BECN1-mediated autophagy. (B) pSERPINB1-Myc or empty vector was transfected into BECN1+/+ and BECN1−/− HEK-293T cells. At 24 hpt, cells were infected with SVA (MOI 0.1) for 24 h. The cells were harvested for western blot with antibodies against VP2, Myc, BECN1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ImageJ was used to quantify the level of protein, and GAPDH was used as a loading control. (C) IBRS-2 cells were transfected with pSERPINB1-Myc or empty vector. At 12 hpt, cells were treated with dimethylsulfoxide or 3MA for 12 h and then cells were infected with SVA. The cells were harvested for western blot analysis. ImageJ was used to quantify the level of protein, and GAPDH was used as a loading control. The experiment was performed three times. Data are expressed as the means ± SEM. ns, not significant; **, P < 0.01, ***, P < 0.001.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Infection, Western Blot, Control

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: Proposed model for SERPINB1-mediated promotion of SVA replication. SERPINB1, a serine protease inhibitor, promotes K48-linked polyubiquitination of IKBKE and degrades IKBKE through the proteasome pathway. The removal of IKBKE by SERPINB1 inhibits the type I IFN signaling via the autophagy pathway to facilitate SVA replication.

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Protease Inhibitor

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: Primers used for plasmid construction

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Plasmid Preparation, Sequencing

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: Primers for truncating the recombinant protein construction

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Recombinant, Sequencing

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: Gene-specific probes for qRT-PCR

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy

doi: 10.1128/jvi.01045-23

Figure Lengend Snippet: Sequences of siRNAs and sgRNAs

Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1-Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Techniques: Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: Establishment and characterization of rat anti-human CLDN10 mAbs. ( A ) Gene structure and protein topology of hCLDN10. Arrowheads reveal transcription start sites, and the antigen region is indicated in red. UTR, untranslated region. ( B ) Amino acid sequences of the C-terminal cytoplasmic domains of hCLDN10 and the corresponding regions of the closely related hCLDN members. Conserved amino acids and the antigen region are shown in light blue and red, respectively. ( C , D ) Immunohistochemical and Western blot analyses showing the specificity of the anti-hCLDN10 mAb (clone #25). 293T cells were transfected with the indicated expression vectors and subjected to analyses using the indicated anti-CLDN10 Abs. Scale bar, 50 μm. ( E ) The complementarity-determining regions (CDRs) of the anti-hCLDN10 mAb (clone #25)

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Immunohistochemical staining, Western Blot, Transfection, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: High CLDN10 expression is associated with poor prognosis in ccRCC patients. ( A ) Confocal images of the indicated proteins in ccRCC tissues. Scale bar, 20 μm. ( B ) Representative immunohistological images showing negative/weak/moderate/strong signal intensity for CLDN10 expression in ccRCC tissues. HE, hematoxylin-eosin. Scale bar, 200 μm. ( C ) Cancer-specific survival in the CLDN10-low and CLDN10-high expression groups of ccRCC patients

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: Relationship between CLDN10 expression and clinicopathological factors in patients with ccRCC

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: Cell surface CLDN10 promotes malignant behavior of the ccRCC cell line 786-O. ( A ) The construct of the hCLDN10A expression vector. EF-1α, elongation factor-1α; IRES, internal ribosome entry site. ( B ) Western blot for the indicated proteins in the revealed 786-O cells. ( C ) Confocal images of CLDN10 in the indicated cells. Scale bar, 20 μm. ( D-F ) Quantitative cell viability, proliferation, and wound healing assays in the indicated cells. The levels are plotted and shown in the histograms (mean ± SD). n = 8, 4, and 18 for D, E, and F, respectively. ( G ) GSEA showing the enrichment of mTOR and PI3K/AKT/mTOR signalings, as well as gene sets of MYC targets in 786-O: CLDN10A cells

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Construct, Expressing, Plasmid Preparation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: CLDN10 forms a complex with LAT1 in 786-O: CLDN10A and 293T + CLDN10A cells. ( A ) Immunoprecipitation-mass spectrometry (IP-MS) analysis revealing that CLDN10, TUBB2A, HLA-H, and LAT1 are potentially associated with CLDN10 in 786-O: CLDN10A cells. ( B , C ) Confocal images of the indicated proteins in the revealed 786-O cells ( B ) and ccRCC tissues ( C ). Yellow arrowheads show colocalization of CLDN10A and LAT1 on cell membranes. Scale bars, 20 μm. ( D ) Three-dimensional structural analysis using PyMOL indicating that the first transmembrane domain of CLDN10A (CLDN10A -TM1) binds to LAT1-TM4. ( E ) IP-IB analysis showing the CLDN10A/LAT1 and CD98/LAT1 complexes in 293T cells. ( F ) IP-IB analysis revealing that the association between CLDN10A∆N and LAT1 is markedly decreased in 293T cells. Transmembrane domains are shown in red. ( G ) IP-IB analysis indicating that LAT1-mTM4 hardly form a complex with CLDN10A in 293T cells. The substituted amino acids are shown in red. IP, immunoprecipitation; IB, immunoblot; HA, hemagglutinin

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: CLDN10–LAT1 signaling promotes ccRCC progression. ( A , E ) Culture conditions for 786-O and ACHN cells (A for B–D, and E for F–H). Cells were grown in the presence or absence of LAT1 siRNA (both #1 and #2) or JPH203. (B, F) Western blot for the indicated proteins in the revealed 786-O and ACHN cells. The protein levels are normalized to the rehybridized β-actin levels, and the relative levels are shown in the histograms. ( C , D , G , H ) Quantitative cell viability and wound healing assays of the indicated 786-O cells. The levels are plotted and shown in the histograms (mean ± SD). n = 8 for C and G, and n = 6 for D and H

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

doi: 10.1186/s12964-024-01964-5

Figure Lengend Snippet: A schema showing the expression and biological functions of CLDN10 in normal kidney and ccRCC tissues. CLDN10 is exclusively localized at tight junctions of TAL and proximal tubule epithelial cells in normal kidney tissues, and contributes to paracellular transport or barrier. On the other hand, CLDN10 is distributed on the whole cell membranes of CLDN-high ccRCC cells, and cooperates with LAT1, resulting in tumor progression. ccRCC, clear cell renal cell carcinoma; PCT, proximal convoluted tubule; PST, proximal straight tubule; TAL, thick ascending limb; T, tumor

Article Snippet: Next, to test the specificity of the anti-CLDN10 mAb (clone #25) and the commercially available rabbit anti-CLDN10 polyclonal antibody (pAb; Affinity Biosciences, AF0133) [ ], 293T cells were transiently transfected with distinct hCLDN expression vectors.

Techniques: Expressing