pimago phosphoprotein detection kit  (Tymora Analytical Operations LLC)

Journal: bioRxiv

Article Title: Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

doi: 10.1101/2025.11.01.685996

Figure Lengend Snippet: (A) Structural snapshots illustrating key interactions of hC5a-hC5aR1 (PDB: 8IA2) and hC5a -d-Arg -hC5aR1 (PDB: 8JZZ). Models are represented in tube helices with hC5a in royal blue, hC5a -d-Arg in lime green, hC5a-bound hC5aR1 in brick-red and hC5a -d-Arg -bound hC5aR1 in dodger-blue. Molecular dynamic simulation (MDS) (lower panel) highlighting the percentage involvement of orthosteric binding residues with hC5a and hC5a -d-Arg . (B-C) cAMP inhibition ( B ) and βarr1 recruitment ( C ) measured downstream of hC5aR1 mutants (R .64 A, R .42 A, D .35 A), rationally designed based on MDS studies. Data represents mean ± SEM (n=3), independent experiments. (D) Cartoon representation comparing helix 8 movement in hC5a-hC5aR1 (8IA2) Vs. hC5a -d-Arg -hC5aR1 (8JZZ) and mC5a-mC5aR1 Vs. mC5a -d-Arg -mC5aR1 structures with respect to NTSR1-GRK2 (PDB: 8JPF) structure, revealing distinct positioning of helix 8 in C5a -d-Arg -bound C5aR1, potentially occluding GRK docking site in the receptor. (E) Structure-based simulation studies comparing C5a and C5a -d-Arg activation of C5aR1 highlighting a key difference in TM7 conformation, measured by the distance between Ile . and Phe . (indicated by black dots) in C5a -d-Arg and C5a-activated mutants. (F) Structural superposition of mC5a and mC5a -d-Arg -bound mC5aR1 highlighting the loss of hydrogen bonds with Ser . and Asn . of TM7 when bound to C5a -d-Arg . (G) Structural superposition of mC5a-mC5aR1 and mC5a -d-Arg -mC5aR1 (left-panel) highlighting the transmembrane regions (TM) undergoing reduced HDX. HDX-MS analysis (right) of mC5aR1 highlighted regions (left) undergoing significant decrease in deuterium uptake upon incubation either with ligand (mC5a/mC5a -d-Arg ) or without ligand (Apo-mC5aR1). Statistical analysis was performed by applying one-way ANOVA followed by Tukey’s multiple comparisons test. (p<0.001***, p<0.01**). (H) NanoBiT-assay showing GRK recruitment downstream to (h/m) C5aR1 in response to (h/m) C5a and C5a -d-Arg .Data represents mean ± SEM (n=3), independent experiments, normalized with lowest ligand concentration considered as 1. (I) Phosphorylation detection via pIMAGO kit (upper panel) and phosphorylation site-specific antibodies (lower panel) to assess the receptor phosphorylation following ligand stimulation. Representative blots and densitometric analysis are shown below (green-mC5a, pink – mC5a -d-Arg , blue – hC5a and maroon – hC5a -d-Arg . Densitometric plots represent mean ± SEM (n = 3-4), independent experiments, with stimulated condition normalized with respect to unstimulated condition which is considered as 100% (p<0.0001****, p=0.0001***, p=0.0013**).

Article Snippet: Agonist-induced phosphorylation downstream of mC5aR1 was measured by using pIMAGO phosphoprotein detection kit from Sigma (Cat. No. 18419), following the manufacturer’s protocol.

Techniques: Binding Assay, Inhibition, Activation Assay, Incubation, Concentration Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

doi: 10.1101/2025.11.01.685996

Figure Lengend Snippet: (A) Structural snapshots illustrating key interactions of hC5a-hC5aR1 (PDB: 8IA2) and hC5a -d-Arg -hC5aR1 (PDB: 8JZZ). Models are represented in tube helices with hC5a in royal blue, hC5a -d-Arg in lime green, hC5a-bound hC5aR1 in brick-red and hC5a -d-Arg -bound hC5aR1 in dodger-blue. Molecular dynamic simulation (MDS) (lower panel) highlighting the percentage involvement of orthosteric binding residues with hC5a and hC5a -d-Arg . (B-C) cAMP inhibition ( B ) and βarr1 recruitment ( C ) measured downstream of hC5aR1 mutants (R .64 A, R .42 A, D .35 A), rationally designed based on MDS studies. Data represents mean ± SEM (n=3), independent experiments. (D) Cartoon representation comparing helix 8 movement in hC5a-hC5aR1 (8IA2) Vs. hC5a -d-Arg -hC5aR1 (8JZZ) and mC5a-mC5aR1 Vs. mC5a -d-Arg -mC5aR1 structures with respect to NTSR1-GRK2 (PDB: 8JPF) structure, revealing distinct positioning of helix 8 in C5a -d-Arg -bound C5aR1, potentially occluding GRK docking site in the receptor. (E) Structure-based simulation studies comparing C5a and C5a -d-Arg activation of C5aR1 highlighting a key difference in TM7 conformation, measured by the distance between Ile . and Phe . (indicated by black dots) in C5a -d-Arg and C5a-activated mutants. (F) Structural superposition of mC5a and mC5a -d-Arg -bound mC5aR1 highlighting the loss of hydrogen bonds with Ser . and Asn . of TM7 when bound to C5a -d-Arg . (G) Structural superposition of mC5a-mC5aR1 and mC5a -d-Arg -mC5aR1 (left-panel) highlighting the transmembrane regions (TM) undergoing reduced HDX. HDX-MS analysis (right) of mC5aR1 highlighted regions (left) undergoing significant decrease in deuterium uptake upon incubation either with ligand (mC5a/mC5a -d-Arg ) or without ligand (Apo-mC5aR1). Statistical analysis was performed by applying one-way ANOVA followed by Tukey’s multiple comparisons test. (p<0.001***, p<0.01**). (H) NanoBiT-assay showing GRK recruitment downstream to (h/m) C5aR1 in response to (h/m) C5a and C5a -d-Arg .Data represents mean ± SEM (n=3), independent experiments, normalized with lowest ligand concentration considered as 1. (I) Phosphorylation detection via pIMAGO kit (upper panel) and phosphorylation site-specific antibodies (lower panel) to assess the receptor phosphorylation following ligand stimulation. Representative blots and densitometric analysis are shown below (green-mC5a, pink – mC5a -d-Arg , blue – hC5a and maroon – hC5a -d-Arg . Densitometric plots represent mean ± SEM (n = 3-4), independent experiments, with stimulated condition normalized with respect to unstimulated condition which is considered as 100% (p<0.0001****, p=0.0001***, p=0.0013**).

Article Snippet: This further translates into inefficient phosphorylation of the C5aR1 as reflected in bulk phosphorylation measured using the pIMAGO assay, and site-specific phosphorylation measured using phospho-site-specific antibodies ( ).

Techniques: Binding Assay, Inhibition, Activation Assay, Incubation, Concentration Assay, Phospho-proteomics