Journal: Frontiers in Endocrinology

Article Title: Mouse Spexin: (II) Functional Role as a Satiety Factor inhibiting Food Intake by Regulatory Actions Within the Hypothalamus

doi: 10.3389/fendo.2021.681647

Figure Lengend Snippet: Primer sequences and PCR conditions for RT-PCR of mouse GALR2/3.

Article Snippet: For Western blot, tissue lysate prepared was subjected to SDS-PAGE and transblotted onto a PVDF membrane followed by immunoblotting using the antibodies for mouse GalR2 and GalR3, respectively (Alomone Labs, 1:8000).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Mouse Spexin: (II) Functional Role as a Satiety Factor inhibiting Food Intake by Regulatory Actions Within the Hypothalamus

doi: 10.3389/fendo.2021.681647

Figure Lengend Snippet: Receptor specificity for SPX inhibition on food consumption in mice. (A) RT-PCR for GalR2 and GalR3 expression in the liver, brain and hypothalamus. Total RNA isolated from target tissues was used for RT-PCR with primers for GalR2 and GalR3, respectively, with GAPDH as internal control and PCR with no template as negative control (“-ve Ctrl”). (B) Western blot of GalR2 and GalR3 expression in the brain and hypothalamus. Tissue lysate was prepared from mouse brain and hypothalamus and subjected to SDS- PAGE and Western blot using the antibody (Ab) for GalR2 and GalR3, respectively, with β actin as loading control. To confirm the specificity of immunoblotting, Western blot was also conducted with antibodies pre-absorbed with the synthetic peptides for GalR2/3 used to raise the antibody provided by the company. (C) GalR2 and GalR3 blockade on the inhibitory effect of SPX on food intake in mice. IP injection of SPX (5 nmol/mouse) was performed at 10:00 am with/without co-treatment of the GalR2 antagonist M871 (50 nmol/mouse) or GalR3 antagonist SNAP (50 nmol/mouse) to test the effect on food consumption occurred during the first hour in dark phase. (D) Effect of GalR2 activation on food consumption in mice. In parallel experiment, IP injection of the GalR2 agonist dN1-Qu (10 nmol/mouse) was conducted with SPX treatment (10 nmol/mouse) as positive control. For the data presented, the groups denoted by different letters represent a significant difference at p < 0.05.

Article Snippet: For Western blot, tissue lysate prepared was subjected to SDS-PAGE and transblotted onto a PVDF membrane followed by immunoblotting using the antibodies for mouse GalR2 and GalR3, respectively (Alomone Labs, 1:8000).

Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Negative Control, Western Blot, SDS Page, Injection, Activation Assay, Positive Control

Journal: Frontiers in Endocrinology

Article Title: Mouse Spexin: (II) Functional Role as a Satiety Factor inhibiting Food Intake by Regulatory Actions Within the Hypothalamus

doi: 10.3389/fendo.2021.681647

Figure Lengend Snippet: Receptor specificity for SPX regulation of hypothalamic expression of orexigenic factors and their receptors. IP injection of SPX (5 nmol/mouse) was performed in mice with/without the co-treatment of the GalR2 antagonist M871 (50 nmol/mouse) or GalR3 antagonist SNAP (50 nmol/mouse). The hypothalamus was harvested one hour later after IP injection. Total RNA was isolated and subjected to real-time PCR for transcript expression of NPY (A) , AgRP (B) , NPY5R (C) and GHSR (D) , respectively with β actin mRNA as internal control. The groups denoted by different letters represent a significant difference at p < 0.05.

Article Snippet: For Western blot, tissue lysate prepared was subjected to SDS-PAGE and transblotted onto a PVDF membrane followed by immunoblotting using the antibodies for mouse GalR2 and GalR3, respectively (Alomone Labs, 1:8000).

Techniques: Expressing, Injection, Isolation, Real-time Polymerase Chain Reaction