Journal: Frontiers in physiology

Article Title: Activation of Orexin System Stimulates CaMKII Expression.

doi: 10.3389/fphys.2021.698185

Figure Lengend Snippet: FIGURE 4 | Orexin-A treatment increases CaMKII subunits’ protein expression as well as CaMKII activation in PC12-OX1R cells. PC12-OX1R cells were incubated with 100 nM orexin-A for the indicated periods. Proteins were separated and probed with CaMKIIa, CaMKIIb, P-CamKII, and β-actin antibodies. (A) Orexin-A treatment increases CaMKII protein expression in a time-dependent manner. (A1) Representative western blots showing CaMKIIa, CaMKIIb, and β-actin in orexin-A treated and control cells. Quantification of CaMKIIa (A2) and CaMKIIb (A3) that has been normalized against β-actin and compared with control, normalized to unity (n = 6, *P < 0.05). (B) Orexin-A treatment results in a time-dependent increase in P-CaMKII level. (B1) representative western blots showing P-CaMKII and β-actin. (B2) Quantification of P-CaMKII that has been normalized against β-actin and compared with control, normalized to unity (n = 6, *P < 0.05, **P < 0.01).

Article Snippet: Antibodies Company Catalog number Source Dilutions for WB Dilution for IF Primary antibodies OX1R Acris Ap31067pu-n Goat / 1:50 OX1R Alomone labs AOR-001 Rabbit / 1:100 NeuN Cell Signaling mAb#24307 Rabbit / 1:50 Iba1 Wako 019-19741 Rabbit / 1:500 GFAP Invitrogen mAb#3670 Mouse / 1:300 Anti-pCaMKII Santa cruz sc-32289 Mouse 1:1000 1:200 CaMKIIa Santa cruz sc-13141 Mouse 1:1000 / CaMKIIb Santa cruz sc-376828 Mouse 1:1000 / β-actin Santa cruz sc-47778 Mouse 1:1000 / Secondary antibodies Anti-goat 488 Invitrogen A32814 Donkey / 1:1000 Anti-rabbit 594 Invitrogen A-21207 Donkey / 1:1000 Anti-rabbit 488 Invitrogen A-21206 Donkey / 1:1000 Anti-mouse 594 Invitrogen A-11005 Goat / 1:1000 Anti-mouse IgG (HRP) Invitrogen PA1-28568 Rabbit 1 :2000 / WB, western blot; IF, immunofluorescence.

Techniques: Expressing, Activation Assay, Incubation, Western Blot, Control