Journal: Journal of Advanced Research

Article Title: Development and characterization of endolysosomal trafficking targeting chimera degraders of α 1A -adrenergic receptor

doi: 10.1016/j.jare.2025.06.014

Figure Lengend Snippet: α 1A -ARs undergo endocytic degradation when stimulated by various agonists, and interact with GASP1 and Beclin 2 during this process. (A) A schematic illustration of the process of agonist-induced postendocytic sorting of GPCRs. (B) Chemical structures of α 1A -AR agonists. (C) Agonist-dependent downregulation of α 1A -AR. α 1A -AR stably transfected HEK293 cells were incubated with agonists at indicated concentrations for 48 h. (D) Quantification of protein levels in C (n = 3 biologically independent experiments). (E, F) HEK293 cells stably expressing Flag-α 1A -AR were lysed and receptors were immunoprecipitated with anti-FLAG antibodies, resolved by SDS/PAGE, and electroblotted. The blot was then incubated with antibodies recognizing GASP1 and HA. Input shows lysates immunoblotted for GASP1 (E) and cGASP (F). (G) Coimmunoprecipitation of endogenous human GASP1 with human HA-Beclin 2 in HEK293 cells transfected with HA-Beclin 2 plasmids. (H) Coimmunoprecipitation of endogenous human GASP1 and Beclin 2 with Flag epitope-tagged α 1A -AR to form a ternary Beclin 2-GASP1-α 1A -AR complex in HEK293 cells stably expressing Flag-α 1A -AR. (I) His-tag labeled GASP1 selectively binds to a GST fusion protein containing the C-terminal tail of α 1A -AR, but not to GST alone. (J) HA-cGASP competed with endogenous GASP1 for binding to α 1A -AR in vivo. In cells overexpressing HA-cGASP, no endogenous GASP1 was detected in receptor immunoprecipitates, but cGASP coimmunoprecipitated with α 1A -AR. (E–J) Similar results were observed in three independent experiments. Data are represented as means ± SD where relevant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. two-sided unpaired t -test.

Article Snippet: Western blot analysis was carried out using the following antibodies after SDS-PAGE was used to separate the eluates: anti-human Beclin 2 (Novus Biologicals; NB110-60982; 1:1000), anti-GASP1 (Proteintech; 20020-1-AP; 1:500), anti-Flag (Proteintech; 66008-4-lg; 1:2000), anti-HA (Proteintech; 51064-2-AP; 1:5000), anti-Bcl-2 (Proteintech; 12789-1-AP; 1:2000).

Techniques: Stable Transfection, Transfection, Incubation, Expressing, Immunoprecipitation, SDS Page, FLAG-tag, Labeling, Binding Assay, In Vivo

Journal: Journal of Advanced Research

Article Title: Development and characterization of endolysosomal trafficking targeting chimera degraders of α 1A -adrenergic receptor

doi: 10.1016/j.jare.2025.06.014

Figure Lengend Snippet: Effects of GASP1 and Beclin 2 on agonist-induced lysosomal transport of α 1A -ARs. (A–C) Effects of Beclin 2 siRNA (B) or cGASP overexpression (C) on biotin-mediated protection of the α 1A -AR after agonist treatment (phenylephrine, 100 μM) in HEK293 cells stably expressing Flag-α 1A -AR. For (A–C), the first lane (100 %) represents total biotinylated GPCR prior to cell-surface biotin stripping; the second lane (strip) represents the remaining biotinylated α 1A -AR after cell-surface stripping; the third, fourth and fifth lanes (10′, 30′, 180′) represent levels of internalized biotinylated receptor with agonist treatment for 10 min, 30 min or 180 min, respectively, after stripping. (D) Quantification of the ratio of α 1A -AR levels at 180 min versus 30 min in each condition shown in A–C (n = 3 biologically independent experiments). Data are represented as means ± SD where relevant. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA with the Tukey’s multiple comparison test. (E) Immunofluorescence analysis of effects of Beclin 2 siRNA or overexpressed cGASP on fate of cell-surface-labeled Flag-α 1A -AR. Cells were fed with anti-Flag antibody, and treated with phenylephrine (100 μM) for 30 or 180 min. Subcellular localization of Flag-α 1A -AR was detected by immunofluorescent staining. Green, Flag- α 1A -AR. Scale bar, 15 μm. (F, G) Fluorescent microscopic imaging of α 1A -AR in HEK293 cells stably expressing Flag-α 1A -AR treated with Beclin 2 siRNA or overexpressed cGASP. Cells were fed with the anti-Flag antibody, treated with phenylephrin (100 μM) for 30 or 90 min, immunostained with anti-EEA1 (F) or anti-LAMP1 (G) primary antibodies, and labeled with fluorescent secondary antibodies. Green, Flag-α 1A -AR; red, EEA1 or LAMP1. Scale bar, 15 μm. (H) Expression of Beclin 2 in HEK293 cells cotransfected with Beclin 2 siRNA and Beclin 2 SSM rescue plasmids. (I, J) Effects of Beclin 2 siRNA and WT-SSM (I) or I80S-SSM (J) plasmid cotransfection on lysosomal degradation of α 1A -AR after agonist treatment (phenylephrine, 100 μM) in HEK293 cells stably expressing Flag-α 1A -AR. (K) Quantification of the ratio of α 1A -AR levels at 180 min versus 30 min in each condition shown in I and J (n = 3 biologically independent experiments). Data are represented as means ± SD where relevant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. two-sided unpaired t -test. For (E–G), similar results were observed in three independent experiments (50 cells analyzed per experiment). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Western blot analysis was carried out using the following antibodies after SDS-PAGE was used to separate the eluates: anti-human Beclin 2 (Novus Biologicals; NB110-60982; 1:1000), anti-GASP1 (Proteintech; 20020-1-AP; 1:500), anti-Flag (Proteintech; 66008-4-lg; 1:2000), anti-HA (Proteintech; 51064-2-AP; 1:5000), anti-Bcl-2 (Proteintech; 12789-1-AP; 1:2000).

Techniques: Over Expression, Stable Transfection, Expressing, Stripping Membranes, Comparison, Immunofluorescence, Labeling, Staining, Imaging, Plasmid Preparation, Cotransfection

Journal: Journal of Advanced Research

Article Title: Development and characterization of endolysosomal trafficking targeting chimera degraders of α 1A -adrenergic receptor

doi: 10.1016/j.jare.2025.06.014

Figure Lengend Snippet: GASP1 and Beclin 2 functions in ETTAC-induced lysosomal degradation of α 1A -AR. (A) Schematic illustration of the ETTAC strategy for GPCR degradation. (B) Chemical structures of ETTACs. (C) K i value was obtained from radio-ligand competitive binding assay (Data are means ± SD, n = 2). (D–F) Immunoblotting analysis of α 1A -AR stably transfected HEK293 cells treated with PMA-19 (D), PMA-37 (E) and PMA-43 (F) at the indicated concentrations (24 h). (G) Immunoblotting analysis of α 1A -AR stably transfected HEK293 cells treated with PMA-37, phenylephrine, PE-Linker, or ML246 (1 μM) for 24 h. (H) Immunoblotting analysis of α 1A -AR stably transfected HEK293 cells treated with 1 μM PMA-37 for 24 h along with 100 nM bafilomycin A1 (pre-treated for 6 h) or 5 nM carfilzomib (co-treated for 24 h). (I) Immunoblotting analysis of α 1A -AR stably transfected HEK293 cells treated with PMA-37 (1 μM, 24 h) under siRNA-mediated knockdown of Beclin 2 (100 pmol, 48 h) or cGASP overexpression (2 ug, 48 h). (J) Effects of Beclin 2 siRNA and indicated plasmid co-transfection on the degradation of the α 1A -AR after PMA-37 treatment (1 μM, 24 h) in HEK293 cells. (K) Coimmunoprecipitation of endogenous human GASP1 and endogenous human Beclin 2 with Flag-human α 1A -AR in HEK293 cells treated with normal medium or normal medium with PMA-37 (1 μM) for 24 h. (L) Real-time calcium influx kinetics measured by Fluo-4 AM fluorescence over 45 s. PC-3 cells were pretreated with 1 μM PMA-37, 1 μM prazosin, or vehicle control for 24 h, followed by stimulation with 100 μM phenylephrine. (M) Quantification of peak calcium influx. (N) Immunoblotting analysis of phosphorylated ERK1/2 and total ERK1/2 in PC-3 cells pretreated for 24 h with 1 μM ETTAC, 1 μM prazosin, or vehicle, followed by stimulation with 100 μM phenylephrine for indicated times. (O) Quantitative densitometric analysis of p-ERK1/2 levels normalized to total ERK1/2 expression. (D–O) Similar results were observed in three independent experiments. Data are represented as means ± SD where relevant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. two-sided unpaired t -test.

Article Snippet: Western blot analysis was carried out using the following antibodies after SDS-PAGE was used to separate the eluates: anti-human Beclin 2 (Novus Biologicals; NB110-60982; 1:1000), anti-GASP1 (Proteintech; 20020-1-AP; 1:500), anti-Flag (Proteintech; 66008-4-lg; 1:2000), anti-HA (Proteintech; 51064-2-AP; 1:5000), anti-Bcl-2 (Proteintech; 12789-1-AP; 1:2000).

Techniques: Competitive Binding Assay, Western Blot, Stable Transfection, Transfection, Knockdown, Over Expression, Plasmid Preparation, Cotransfection, Fluorescence, Control, Expressing

Journal: Viruses

Article Title: Reduced-Beclin1-Expressing Mice Infected with Zika-R103451 and Viral-Associated Pathology during Pregnancy

doi: 10.3390/v12060608

Figure Lengend Snippet: ZIKV infection in Becn1 +/+ and Becn1 +/− pregnant dams. ( A ) Schematic diagram illustrating Zika virus (ZIKV)-infection in timed-pregnant dams. Prior to viral infection, pregnant dams received the antibody MAR1-5A3 at 2 mg/animal via intraperitoneal (ip) route at gestational day 8 followed by subcutaneous (sc) infection with ZIKV at 10 3 plaque-forming unit (PFU) in 50 µL of PBS or mock (PBS) injection at gestational day E9. ( B ) Representative Western Blots probed with antibodies against several autophagy proteins, and B-actin was used as an internal control. Adult Becn1 +/+ and Becn1 +/− brains were removed postmortem and minced according to the Materials and Methods. ( C ) Densitometric analysis using image J indicate the levels of p62, Beclin1, ATG5, LC3-I and LC3-II in brains of adult Becn1 +/+ (black bar) and Becn1 +/− (brown bar) mice. The error bars show mean ± SEM for N = 3 animals per treatment. The data were analyzed using GraphPad Prism and two-way analysis of variance (ANOVA) followed by Tukey’s test. * p < 0.05 and ** p < 0.01 vs. Becn1 +/+ . ( D ) Weight gain, expressed in grams, was measured using an analytical balance at gestation day 0 and throughout gestation period, at 3-day intervals. ( E ) Percent survival rate in pregnant dams infected with ZIKV or mock (PBS) was calculated by dividing the total number of live animals by the number of live + dead animals X 100. ( F ) Viral RNA detected in serum collected from ZIKV-infected dams on E13. ( G ) Viral RNA detected in organs removed postmortem from ZIKV-infected dams on E17. ( D – G ) Error bars show mean ± SEM for N = 5–8 animals per treatment. The data were analyzed using GraphPad Prism and two-way ANOVA followed by Tukey’s test. * p < 0.05 vs. Becn1 +/+ . ( F , G ) Viral RNA equivalent is expressed on a log10 scale after comparison with a standard curve produced using serial 10-fold dilutions of ZIKV RNA from known quantities of infectious virus.

Article Snippet: Immunoblots were labeled with primary antibodies against Beclin1 (Catalog# NB500–249), ATG5 (Catalog# NB110-53818), LC3-B (Catalog# NB600–1384) and P62/SQSTM1 (Catalog# NBP1–48320) purchased from Novus Biologicals (Centennial, CO, USA). β-actin (Catalog# sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as internal control.

Techniques: Infection, Virus, Injection, Western Blot, Control, Mass Measurement, Comparison, Produced

Journal: Viruses

Article Title: Reduced-Beclin1-Expressing Mice Infected with Zika-R103451 and Viral-Associated Pathology during Pregnancy

doi: 10.3390/v12060608

Figure Lengend Snippet: Potential link between ZIKV proteins and Beclin1 protein. ( A – D ) The secretions of pro-inflammatory molecules were detected in glial supernatants exposed to 50 nM of viral proteins after 8, 24- and 96-h by ELISA. Becn1 +/+ glia (black bar) and Becn1 +/− glia (brown bar). The data were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05 vs. respective media control, # p < 0.05 vs. Becn1 +/+ .

Article Snippet: Immunoblots were labeled with primary antibodies against Beclin1 (Catalog# NB500–249), ATG5 (Catalog# NB110-53818), LC3-B (Catalog# NB600–1384) and P62/SQSTM1 (Catalog# NBP1–48320) purchased from Novus Biologicals (Centennial, CO, USA). β-actin (Catalog# sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as internal control.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control