f2rl3 par4  (Addgene inc)

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: Antibodies to TP (27159-1-AP), P2Y1 (67654-1-AP), and PAR4 (25306-1-AP) were purchased from Proteintech Group, Inc (Rosemont, IL).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: To investigate how α1 affects platelet GPCR signaling function and its interaction with certain platelet GPCRs, we transfected COS-7 cells (ATCC, CRL-1651) with plasmids encoding P2Y12 (item#66471, Addgene), F2RL3 (PAR4) (item #66279), TP (item #66517), or A2BR (item#37202) using LipofectamineTM 3000 Transfection Reagent (L3000015, ThermoFisher Scientific).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control

Journal: British Journal of Pharmacology

Article Title: Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post‐inflammatory rat model for irritable bowel syndrome

doi: 10.1111/bph.14396

Figure Lengend Snippet: TaqMan primers used for qPCR analysis of colonic and DRG samples

Article Snippet: Rabbit anti‐PAR4 was purchased from Alomone Labs. Rabbit anti‐PAR2 was obtained from Santa Cruz Biotechnology.

Techniques:

Journal: British Journal of Pharmacology

Article Title: Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post‐inflammatory rat model for irritable bowel syndrome

doi: 10.1111/bph.14396

Figure Lengend Snippet: Immunohistochemical localization of PAR2, PAR4 and TRPA1 channels in sensory nerve fibres of the distal colon. (A–C) Representative images showing co‐localization of PAR2 (red) in CGRP‐immunopositive nerve fibres (green). (D–F) Representative images showing the presence of PAR4 immunoreactivity (red) in the colonic epithelium and in enteric nerve plexuses (arrowheads) but not in the CGRP‐immunoreactive nerve fibre population (inset). (G–I) Representative images showing co‐localization of TRPA1 protein (red) in CGRP‐immunopositive nerve fibres (green).

Article Snippet: Rabbit anti‐PAR4 was purchased from Alomone Labs. Rabbit anti‐PAR2 was obtained from Santa Cruz Biotechnology.

Techniques: Immunohistochemical staining

Journal: British Journal of Pharmacology

Article Title: Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post‐inflammatory rat model for irritable bowel syndrome

doi: 10.1111/bph.14396

Figure Lengend Snippet: Relative mRNA expression of PARs and TRP channels in samples from colons, DRG T13‐L2 and DRG L6‐S1

Article Snippet: Rabbit anti‐PAR4 was purchased from Alomone Labs. Rabbit anti‐PAR2 was obtained from Santa Cruz Biotechnology.

Techniques: Expressing