β2ar  (Bioss)

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) GαsBP1 and GαsBP2 block adenylyl cyclase (AC) activity stimulated by Gαs, but not by forskolin. Purified catalytic domains of AC were incubated with Gαs (0.1 μM) or Forskolin (10 μM) in the presence of the indicated concentrations GαsBP1 (green) or GαsBP2 (orange). IC 50 values were determined from curve fits. Mean ± SEM. (n = 3 and 2 independent experiments for Gαs and forskolin, respectively). (B, C) GαsBP2, but not GαsBP1, binds specifically to Gαs but not to α-subunits of other G protein families. Purified GST-fused peptides immobilized on glutathione-agarose beads were incubated with lysates of HEK293T cells expressing constitutively active mutants of the indicated G proteins fused to YFP. Bead-bound proteins were detected by Ponceau S staining or immunoblotting (IB). Representative of three independent experiments. (C) Subcellular localization of cytosolic GαsBP2 (cyto-GαsBP2) and membrane-anchored GαsBP2 (mem-GαsBP2). Confocal microscopy images of HEK293T cells transfected with GαsBP2 constructs as in the cAMP experiments and stained as indicated. Scale bars = 10 μm. (E) mem-GαsBP2, but not cyto-GαsBP2, blocks GPCR-stimulated AC activity in cells, whereas the GαsBP2 W11A mutants have no effect. Diagram on the top left represents the mechanism of GPCR-G s -AC signaling interrogated by GαsBP2 probes. Kinetic traces on the right are luminescence-based cAMP measurements were carried out in HEK293T cells expressing the indicated constructs. Mean ± S.E.M. (n=4 independent experiments). A representative immunoblotting result confirming the expression of GαsBP2 constructs is shown on the bottom left. (F) mem-GαsBP2 dose-dependently blocks GPCR-stimulated but not forskolin-stimulated AC activity in cells, whereas the GαsBP2 W11A mutant has no effect. Diagram on the left represents the mechanisms AC activation interrogated by GαsBP2 probes. Graphs in the middle correspond to luminescence-based cAMP measurements in HEK293T cells transfected with the indicated amounts of mem-GαsBP2 plasmid after stimulation with isoproterenol (100 nM) or forskolin (10 μM). Mean ± S.E.M. (n=3 independent experiments). A representative immunoblotting result confirming the expression of GαsBP2 is shown on the right.

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Activation Assay, Blocking Assay, Activity Assay, Purification, Incubation, Expressing, Staining, Western Blot, Membrane, Confocal, Microscopy, Transfection, Construct, Mutagenesis, Plasmid Preparation

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) Specific inhibition of Gαs-dependent cAMP production emanating from endosomes by subcellular targeting of GαsBP2. Diagram on the top left depicts the two-component cAMP response mechanism by PTH1R upon PTH 1-34 stimulation, where an early cAMP response at the plasma membrane is followed by sustained cAMP response originating from endosomes. Graphs with kinetic traces correspond to real-time FRET-based cAMP measurements carried out in HEK293T cells transfected with mem-GαsBP2 or endosome-targeted GαsBP2 (endo-GαsBP2) upon brief stimulation with PTH 1-34 (1 min, indicated by the horizontal black line). mem-GαsBP2 leads to a generalized dampening of the cAMP response, whereas endo-GαsBP2 primarily reduces the sustained component of the cAMP response after agonist washout. Scatter plots on the bottom right correspond to the quantification of the early and sustained components of the cAMP responses from the kinetic curves. Mean ± S.E.M. (n=4-9 independent experiments). Statistical testing by unpaired, two-tailed Student’s t-test. Confocal microscopy images of immunostained HEk293T cells on the bottom left confirm the localization of endo-GαsBP2 at intracellular membranes positive for the endosomal marker EEA1. Scale bars = 10 μm. (B) Specific inhibition of Gαs-induced gene expression by subcellular targeting of GαsBP2 to endosomes. Diagrams on the top depict different scenarios of Gαs-mediated regulation of gene expression triggered by β2AR depending on the subcellular localization of GαsBP2. Bottom, Bar graphs on the bottom correspond to relative mRNA levels of PCK1 , FOS , or DUSP1 in HEK293T cells transfected with mem-GαsBP2, endo-GαsBP2, or cyto-GαsBP2 in the presence or absence of isoproterenol stimulation. Mean ± S.E.M. (n=7 independent experiments). Statistical testing by one-way ANOVA corrected for multiple comparisons (Dunnett). Bottom right, representative immunoblotting result confirming the expression of GαsBP2 constructs.

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Inhibition, Clinical Proteomics, Membrane, Transfection, Two Tailed Test, Confocal, Microscopy, Marker, Gene Expression, Western Blot, Expressing, Construct

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) mem-GαsBP2 does not affect Gαs-Gβγ association prior to GPCR stimulation, but accelerates Gβγ binding to Gαs after GPCR signal termination. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and different amounts of mem-GαsBP2 WT or mem-GαsBP2 W11A, as indicated. Changes in BRET before β2AR stimulation compared to the condition without GαsBP2 (“ΔBRET (no GαsBP2)”) are shown in the graphs on the left, and real-time changes in BRET upon isoproterenol (agonist)/ alprenolol (antagonist) treatment compared to the unstimulated baseline (“ΔBRET (baseline)”) are shown in the graphs on the right. Mean ± S.E.M. (n=4-5 independent experiments). Immunoblots on the right correspond to representative results of experiments confirming the expression of mem-GαsBP2 and BRET sensor components is shown of the right. (B) mem-GαsBP2 does not affect the efficacy or potency of isoproterenol on Gβγ responses. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and the indicated amounts of mem-GαsBP2 were stimulated with the indicated concentrations of isoproterenol. The changes in BRET upon isoproterenol stimulation compared to the unstimulated condition (“ΔBRET·10 3 (no agonist)”) are shown on the left graph, the maximal response (“maximum ΔBRET ·10 3 ”) is shown on the middle graph, and the negative logarithm of half-maximal effective concentration (pEC 50 ) is shown on the right graph. Mean ± S.E.M. (n=3 independent experiments). ns, p>0.05 by one-way ANOVA corrected for multiple comparisons (Dunnett). (C) cyto-GαsBP2 accelerates Gβγ binding to Gαs after GPCR signal termination, whereas the GαsBP2 W11A mutant has no effect. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes Real-time changes in BRET compared to the unstimulated baseline (“ΔBRET (baseline)”) were measured in HEK293T cells expressing a BRET-based biosensor for free Gβγ co-transfected with 500 ng of plasmid DNA for cyto-GαsBP2 WT, mem-GαsBP2 WT, or their corresponding W11A mutants, upon isoproterenol (agonist)/ alprenolol (antagonist) treatment. Mean ± S.E.M. (n=4 independent experiments). Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs and BRET sensor components is shown on the right.

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Binding Assay, Bioluminescence Resonance Energy Transfer, Expressing, Western Blot, Concentration Assay, Mutagenesis, Transfection, Plasmid Preparation, Construct

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) endo-GαsBP2 minimally reduces GPCR-stimulated AC activity in cells, whereas mem-GαsBP2 efficiently blocks it. Diagram on the top left corresponds the mechanism of cAMP regulation investigated with GαsBP2 probes. Graphs with kinetic traces on the top right correspond to luminescence-based cAMP measurements in HEK293T cells expressing 100 ng mem-GαsBP2 or 250 ng endo-GαsBP2 stimulated with isoproterenol as indicated. Mean ± S.E.M. (n=4 independent experiments). The bar graph on the bottom left corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n=4 independent experiments); ns, p>0.05, **p<0.01 by one-way ANOVA corrected for multiple comparisons (Dunnett). Immunoblots on the bottom right correspond to representative results of experiments confirming the expression of GαsBP2 constructs. The kinetic curve of the cAMP response from cells expressing mem-GαsBP2 is reproduced from Fig. 2E . (B) endo-GαsBP2 efficiently blocks the GPCR-stimulated ERK phosphorylation. Diagram on the left corresponds the mechanism of ERK1/2 regulation investigated with GαsBP2 probes. ERK1/2 phosphorylation was detected in HEK293T cells expressing endo-GαsBP2 or mem-GαsBP2 were stimulated (or not) with 10 μM isoproterenol for 5min, as indicated. A representative immunoblotting result is shown in the middle and the quantification of ERK1/2 phosphorylation is shown on the bar graph on the right. Mean ± S.E.M. (n=5 independent experiments). *p<0.05 by one-way ANOVA corrected for multiple comparisons (Dunnett).

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Western Blot, Construct

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) Design of different plasma membrane-anchored GαsBP2 constructs. HEK293T cells transfected with GαsBP2-mem constructs were stained as indicated and visualized by confocal fluorescence microscopy. Scale bars = 10 μm. (B) GαsBP2-mem blocks the isoproterenol-stimulated cAMP response as efficiently as mem-GαsBP2. Diagram on the top left corresponds the mechanism of cAMP regulation investigated with GαsBP2 probes. Graphs with kinetic traces on the bottom correspond to luminescence-based cAMP measurements in HEK293T cells expressing the indicated constructs. Mean ± S.E.M. (n=3 independent experiments). Immunoblots on the top right correspond to representative results of experiments confirming the expression of GαsBP2 constructs. (C) GαsBP2-mem blocks the isoproterenol-induced gene transcription as efficiently as mem-GαsBP2. Bar graphs on the bottom correspond to relative mRNA levels of PCK1 or FOS in HEK293T cells transfected with GαsBP2-mem or mem-GαsBP2 in the presence or absence of isoproterenol stimulation, as indicated. Mean ± S.E.M. (n=4 independent experiments); ns, p>0.05, *p<0.05, by one-way ANOVA corrected for multiple comparisons (Dunnett). (D) Diagram on the top shows two potential scenarios of Gαs-mediated regulation of gene expression triggered by β2AR inhibited. Bar graphs on the bottom correspond to relative mRNA levels of PCK1 , FOS , or DUSP1 in HEK293T cells transfected with mem-GαsBP2, endo-GαsBP2, or both constructs simultaneously in the presence or absence of isoproterenol stimulation, as indicated. Mean ± S.E.M. (n=4 independent experiments); ns, p>0.05, *p<0.05, **p<0.01 by one-way ANOVA corrected for multiple comparisons (Dunnett).

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Inhibition, Construct, Clinical Proteomics, Membrane, Transfection, Staining, Confocal, Fluorescence, Microscopy, Expressing, Western Blot, Gene Expression

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) GαsBP2 does not accelerate GTP hydrolysis by Gαs R201C. Single-turnover GTPase activity was measured with [γ- 33 P]GTP-loaded Gαs R201C in the absence (Gαs only, 1% DMSO, grey) or presence GαsBP2 (orange) by quantifying the release of 33 P[Pi] at 21 °C. Mean ± S.E.M. (n=3 independent experiments). (B) Gβγ remains dissociated from Gαs R201C after GPCR signal termination both in the presence and absence of mem-GαsBP2. Diagram on the top left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs on the left correspond to real-time changes in BRET relative to unstimulated cells expressing Gαs WT alone (“ΔBRET (Gαs WT)”) in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and Gαs R201C ( top graph ) or Gαs WT ( bottom graph ), with or without mem-GαsBP2 expression. After isoproterenol stimulation, cells were treated with alprenolol or buffer as indicated. Mean ± S.E.M. (n=4 independent experiments). Immunoblots on the bottom left right correspond to representative results of experiments confirming the expression of mem-GαsBP2, G proteins, and BRET sensor components. (C) mem-GαsBP2 promotes the association of Gαs Q227L with Gβγ. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ, Gαs Q227L, and different amounts of mem-GαsBP2 WT (orange) or mem-GαsBP2 W11A (blue) as indicated. Open circles correspond to controls in which Gαs Q227L was replaced by Gαs WT in the presence of mem-GαsBP2 WT. Changes in BRET (ΔBRET) relative to cells expressing Gαs WT in the absence of mem-GαsBP2 (“ΔBRET (Gαs WT)”) are shown in the graphs. Mean ± S.E.M. (n=4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of mem-GαsBP2, G proteins, and BRET sensor components are shown on the right. (D) mem-GαsBP2, but not cyto-GαsBP2, recruits Gαs Q227L to the plasma membrane. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a plasma membrane-anchored nanoluciferase (mas-Nluc) and Venus-fused Gαs WT or Q227L in the absence or presence of mem-GαsBP2 or cyto-GαsBP2. Changes in BRET relative to cells expressing Gαs WT alone (“ΔBRET·10 3 (Gαs WT)”) are shown in the graph. Mean ± S.E.M. (n=3 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, G proteins variants, and mas-Nluc. (E) As opposed to mem-GαsBP2, cyto-GαsBP2 does not promote the association of Gαs Q227L with Gβγ. Graphs on the left correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ, Gαs WT or Q227L, and the indicated GαsBP2 constructs. Changes in BRET (ΔBRET) relative to cells expressing Gαs WT in the absence of mem-GαsBP2 (“ΔBRET (Gαs WT)”) are shown in the graph. Mean ± S.E.M. (n=4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, G proteins, and BRET sensor components. (F) Proposed model for the mechanism by which mem-GαsBP2 sequesters Gαs Q227L into an inactive complex with Gβγ. Upon recruitment of Gαs Q227L from the cytosol to the plasma membrane by mem-GαsBP2 (red), but not cyto-GαsBP2 (green), concentration of the G protein favors its association with membrane-resident Gβγ.

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Activity Assay, Clinical Proteomics, Membrane, Bioluminescence Resonance Energy Transfer, Expressing, Western Blot, Construct, Concentration Assay

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) Gβγ facilitates the association of Gαs with the plasma membrane. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond BRET measurements in HEK293T cells co-expressing plasma membrane anchored Venus (Venus-KRas), nanoluciferase-fused Gαs (Gαs-Nluc), and different amounts of Gβγ, as indicated, in the presence or absence of 100 nM isoproterenol stimulation as indicated. Results are presented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n=4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of Venus-KRas, Gαs-Nluc, and Gβγ are shown on the right. (B) mem-GαsBP2 prevents the translocation of Gαs from membranes to the cytosol upon the GPCR stimulation. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond BRET measurements in HEK293T cells co-expressing plasma membrane anchored Venus (Venus-KRas), nanoluciferase fused Gαs (Gαs-Nluc), and mem-GαsBP2 WT or mem-GαsBP2 W11A, as indicated. Changes in BRET compared to the unstimulated baseline (“ΔBRET·10 3 (baseline)”) are shown in the kinetic curves on the right graph, whereas the bar graph on the right corresponds to the quantification of ΔBRET before or after isoproterenol stimulation for 10 min. Mean ± S.E.M. (n=4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of Venus-KRas, Gαs-Nluc, and mem-GαsBP2 is shown on the right.

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Bioluminescence Resonance Energy Transfer, Expressing, Western Blot

Journal: Nature chemical biology

Article Title: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling

doi: 10.1038/s41589-025-02138-1

Figure Lengend Snippet: (A) Diagram depicting the G protein dependent effects investigated with α -sintide (B) α-sintide specifically inhibits Gαs activation. Graphs with kinetic traces on the top correspond to BRET measurements in HEK293T cells co-expressing the indicated combination of GPCR and ONE-GO biosensor after preincubation with 100 μM α-sintide WT (orange), 100 μM α-sintide W11A (green), or 1% (v:v) DMSO (grey) for 2 hrs. Data is presented as real-time changes in BRET relative to the unstimulated baseline (“ΔBRET·10 3 (baseline)”). Bar graphs on the bottom display the maximal changes in BRET after agonist stimulation relative to the unstimulated baseline (“Max ΔBRET·10 3 ”). Mean ± S.E.M. (n=3-4 independent experiments). (C) α-sintide has no effect on the basal activity of any of the G proteins investigated. BRET was measured in HEK293T cells as in (A) but without stimulation and results are represented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n=3-4 independent experiments).

Article Snippet: Plasmids encoding human β2AR (pcDNA3-Flag-beta-2-adrenergic receptor, #14697) 100 and human PAR1 (pBJ-FLAG-hPAR1 #53226) 89 were obtained from Addgene.

Techniques: Activation Assay, Expressing, Activity Assay, Bioluminescence Resonance Energy Transfer

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: (a & b) Representative I sc tracings of CFTR-dependent short-circuit currents ( I sc ) were measured in Ussing chambers. (c) Representative Western blot images of total cellular CFTR and β2AR expression levels in treated dBCi cells after Ussing chamber analyses in ( a ). (d) Densitometric analyses showing B[a]P-induced reduction of both β2AR and CFTR expression in a dose-dependent manner. (e) The Pearson correlation coefficient (r) showing a significant positive relationship between B[a]P-inhibited β2AR and CFTR expression. All data were obtained from three independent experiments. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P treatments to the DMSO control; * p < 0.01.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: Both apical and basolateral sides of polarized 16HBE14o-cells (ALI for 8-10 days) were incubated with different concentrations of B[a]P. Treated media from the apical sides were removed after 6-hr incubation, and cell treatment continued further for 18 hrs under ALI conditions. ( a ) Representative I sc tracings of CFTR-dependent short-circuit currents (I sc ) measured in Ussing chambers as the changes in response to Amiloride, IBMX/Forskolin, and CFTRinh-172. (b) Summary of changes in I sc is shown. Data represent the mean of three independent experiments, and each experiment was performed in duplication (n = 3). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P treatments to the DMSO control; * p < 0.05; ** p < 0.01. ( c ) Representative Western blot images of β2AR and CFTR expression levels in treated cells after Ussing chamber analyses in ( a ).

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Incubation, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: Cells were treated with various concentrations of PM2.5 for 24 hr. ( a ) Cell viability was assessed with MTT assay. ( b ) IL-6 and IL-8 from the basolateral sides. ( c ) Representative Western blot images showing CFTR, β2AR, phospho-AKT (Ser473), AKT, phospho-NF-κB p65 (Ser536), NF-κB p65, and TRPC6 expression. All data are presented as the mean ± SD (n = 4). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P or PM 2.5 treatments to the DMSO control; *, p < 0.05; ¥, p < 0.01; NS, no significance.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: MTT Assay, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: Both apical and basolateral sides of polarized 16HBE14o-cells (ALI for 8-10 days) were incubated with different concentrations of PM 2.5. Treated media from the apical sides were removed after 6-hr incubation, and cell treatment continued further for 18 hrs under ALI conditions. ( a ) Representative I sc tracings of CFTR-dependent short-circuit currents (I sc ) measured in Ussing chambers as the changes in response to Amiloride, IBMX/Forskolin, and CFTRinh-172. (b) Summary of changes in I sc is shown. Data represent the mean of three independent experiments, and each experiment was performed in duplication (n = 6). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P treatments to the DMSO control; ¥, p < 0.05; *, p < 0.01. ( c ) Representative Western blot images of β2AR and CFTR expression levels in treated cells after Ussing chamber analyses in ( a). All Western analyses represent the results of three independent experiments, and β-actin was used for equal loading of protein.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Incubation, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: (a) Representative Western blot images of cell surface CFTR and β2AR expression in cells treated with DMSO or various concentrations of B[a]P. (b & c) Recycling assay of endocytosis CFTR and β2AR were analyzed by Western blot. Representative Western blot images of biotinylated CFTR and β2AR expression are shown. Cell samples treated without or with GSH ( lanes 1 and 2 ) were used as positive controls for biotinylation and GSH stripping processes, respectively. (d) A representative Western blot image showing ubiquitination of CFTR immunoprecipitates from cell lysates treated with different concentrations of B[a]P in the presence of 20 µM MG 132. All Western blot analyses represent the results of three independent experiments.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Western Blot, Expressing, Stripping Membranes, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ( a ) Fluo-4-AM fluorescence intensity traces obtained 24 hours after treatment with B[a]P and stimulated with assay buffer containing 1 mM Ca +2 . ( b ) Corresponding histograms summarize the mean changes in RFU (F 1 – F 0 ) after stimulation by 1 mM Ca +2 . ( c & d ) Representative Western blot images of β2AR and TRPC6 expression levels in treated cell in ( a ) and respective densitometric analyses. ( e ) The Pearson correlation coefficient (r) showing a significant negative relationship between β2AR and TRPC6 expression caused by B[a]P treatment. ( f ) Fluo-4AM fluorescence intensity traces in 16HBE14o-cells were obtained before and after addition of 2 mM Ca +2 in the presence or absence of 5µM BI 749327. ( g ) Corresponding histograms summarize the mean changes in RFU (F 1 – F 0 ) after stimulation by 2 mM Ca +2 . ( h ) Representative western blotting analyses of CFTR expression in corresponding treated cells. All data represent the mean of three independent experiments. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P ± BI 749327 treatments to the DMSO control; ¥, p<0.05, * p < 0.01.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Fluorescence, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ( a ) After 24 hr treatment, fluorescence intensity traces obtained using the fluo-4 AM when DMSO- or PM 2.5-treated 16HBE14o-cellswere stimulated with assay buffer containing 2 mM Ca. Each trace is the mean of three independent experiments with standard deviations (vertical bars). ( b ) Corresponding histograms summarize the mean area under the curve (AUC) after stimulation by 2 mM Ca. ( c ) Representative Western blot images of CFTR, β2AR and TRPC6 expression levels in treated cell in ( a ). All data were obtained from four independent experiments. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the PM 2.5 treatments to the DMSO control; ¥, p < 0.05; *, p < 0.01.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Fluorescence, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ( a ) The lung parenchyma from two ferrets were sliced into several sections as shown. Arrows indicate the tracheobronchial tubes. All tissue sections were incubating with DMSO (control), 10 µM B[a]P, or 100 ng/mL PM 2.5 in Pneumacult-EX Plus Basal media for 24 hrs. ( b ) IL 6 secretion was determined using the ferret ELISA kit. Data represent the mean of 6 independent sections. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P or PM 2.5 treatments to the DMSO control; ¥, p < 0.05 (n = 6). ( c ) Representative western blotting images show reduction of CFTR, and β2AR and elevation of TRPC6 expression in explants under these treatments.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: PM 2.5 or the PM 2.5 toxin B[a]P initially acts as an irreversible β2AR agonist to hyperactivate the PI3K signaling pathway. PLC, acting on PIP2, releases DAG. DAG activates the TRPC6 channel to bring extracellular Ca +2 into the cell. Increased Ca +2 is taken up by mitochondria, which release Reactive Oxygen Species (ROS) and contribute to oxidative stress. BI 749327 can block TRPC6 channel activity and suppress oxidative stress. Meanwhile, hyper-activation of β2AR leads to endosomal β2AR loss, followed by endosomal loss of CFTR (-). Loss of CFTR results in (i) activation of TRADD and downstream activation of IKKαβ to drive NFκB; and (ii) further activation of CFTR-bound TRPC6. NFκB enters the nucleus and drives expression of proinflammatory cytokines IL8 and IL6. Both TRADD and PI3K pathways may be required for optimal NFκB activation, because LY294002 can block inflammation.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Blocking Assay, Activity Assay, Activation Assay, Expressing