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: Human Molecular Genetics

Article Title: PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

doi: 10.1093/hmg/ddae051

Figure Lengend Snippet: Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in liver. (A and B) Western blot analysis of PAH, ubiquitin and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in liver samples from 12-week-old mice. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Statistical analysis performed by unpaired t test ( * P < 0.05, ** P < 0.01, ( *** P < 0.001, **** P < 0.0001, ns, not significant). Data are presented as mean ± SEM.

Article Snippet: For the analysis of total proteins , membranes were blocked for 1 h with 5% nonfat milk, in 0.1% TBS-tween and incubated overnight with the corresponding primary antibody: PAH (1:1000, Santa Cruz Biotechnology, sc-271 258), Ubiquitin (1:1000, Santa Cruz Biotechnology, sc-8017), Hsp70 (1:1000, Novus Biological, NB110-61582), DNAJC12 (1/1000, Abcam, ab167425), LC3B (1:1000, Cell Signaling Technology, #2775), LAMP1 (1:1000, Cell Signaling Technology, #3243) and p62/SQSTM1 (1:5000, Novus biologicals, H00008878-M01).

Techniques: Variant Assay, Western Blot, Ubiquitin Proteomics, Control

Journal: Human Molecular Genetics

Article Title: PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

doi: 10.1093/hmg/ddae051

Figure Lengend Snippet: Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in HepG2 cells. (A and B) Representative western blot analysis of PAH and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in HepG2 cells. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Data are presented as mean ± SD of two independent experiment. (D) PAH levels detected by western blot in HepG2 cells 72 h after transfection with 2 μg of the plasmid coding for DNAJC12. GAPDH was used as loading controls.

Article Snippet: For the analysis of total proteins , membranes were blocked for 1 h with 5% nonfat milk, in 0.1% TBS-tween and incubated overnight with the corresponding primary antibody: PAH (1:1000, Santa Cruz Biotechnology, sc-271 258), Ubiquitin (1:1000, Santa Cruz Biotechnology, sc-8017), Hsp70 (1:1000, Novus Biological, NB110-61582), DNAJC12 (1/1000, Abcam, ab167425), LC3B (1:1000, Cell Signaling Technology, #2775), LAMP1 (1:1000, Cell Signaling Technology, #3243) and p62/SQSTM1 (1:5000, Novus biologicals, H00008878-M01).

Techniques: Variant Assay, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Open Biology

Article Title: SLMAP3 is crucial for organogenesis through mechanisms involving primary cilia formation

doi: 10.1098/rsob.240206

Figure Lengend Snippet: Primary antibodies used for western blot analysis.

Article Snippet: STRN3/SG2NA (S68) , Novus Biologicals , NBP74572 , mouse , 1:500.

Techniques: Western Blot