anti-calstabin2 (fkbp12 c-19)  (Santa Cruz Biotechnology)

Journal: Alzheimer's & Dementia

Article Title: Alzheimer's‐like signaling in brains of COVID‐19 patients

doi: 10.1002/alz.12558

Figure Lengend Snippet: Dysregulation of calcium‐handling proteins in COVID‐19 brains. A, Western blots depicting ryanodine receptor 2 (RyR2) oxidation, protein kinase A (PKA) phosphorylation, and calstabin2 or NADPH oxidase 2 (NOX2) bound to the channel from brain (CB, cerebellum; Ctx, cortex) lysates. B, Bar graphs quantifying DNP/RyR2, pS2808/RyR2, and calstabin2 and NOX2 bound to the channel from the Western blots. Data are mean ± standard deviation (SD). * P < .05 control versus COVID‐19; # P < .05 COVID‐19 versus COVID‐19+ARM210. C, 3 [H]ryanodine binding from immunoprecipitated RyR2. Bar graphs show ryanodine binding at 150 nM Ca 2+ as a percent of maximum binding (Ca2+ = 20 μM). Data are mean ± SD. * P < .05 control versus COVID‐19; # P < .05 COVID‐19 versus COVID‐19+ARM210. D, Western blots showing the levels of glutamate carboxypeptidase 2 (GCPII), calbindin, and GAPDH loading control in brain (Ctx, CB). E, Bar graphs quantifying GCPII/GAPDH and calbindin/GAPDH from the western blots. Data are mean ± SD. * P < .05 control versus COVID‐19

Article Snippet: Immunoblots were developed using the following primary antibodies: anti‐RyR2 (Affinity BioReagents, 1:2500), anti‐phospho‐RyR‐Ser(pS)‐2808 (Affinity BioReagents 1:1000), anti‐ calstabin2 (FKBP12 C‐19, Santa Cruz Biotechnology, Inc., 1:2500), and anti‐NOX2 (Abcam, 1:1000).

Techniques: Western Blot, Phospho-proteomics, Standard Deviation, Control, Binding Assay, Immunoprecipitation

Journal: Alzheimer's & Dementia

Article Title: Alzheimer's‐like signaling in brains of COVID‐19 patients

doi: 10.1002/alz.12558

Figure Lengend Snippet: SARS‐CoV‐2 infection results in leaky ryanodine receptor 2 (RyR2) that may contribute to cardiac, pulmonary, and cognitive dysfunction. SARS‐CoV‐2 infection targets cells via the angiotensin‐converting enzyme 2 (ACE2) receptor, inducing inflammasome stress response/activation of stress signaling pathways. This results in increased transforming growth factor‐β (TGF‐β) signaling, which activates SMAD3 (pSMAD) and increases NADPH oxidase 2 (NOX2) expression and the amount of NOX2 associated with RyR2. Increased NOX2 activity at RyR2 oxidizes the channel, causing calstabin2 depletion from the channel macromolecular complex, destabilization of the closed state, and ER/SR calcium leak that is known to contribute to cardiac dysfunction, ⁵⁵ arrhythmias, ⁶¹ pulmonary insufficiency, ²³ , ²⁵ and cognitive and behavioral abnormalities associated with neurodegenreation. ²⁴ , ²⁶ Decreased calbindin in COVID‐19 may render brain more susceptible to tau pathology. Rycal drugs fix the RyR2 channel leak by restoring calstabin2 binding and stabilizing the channel closed state. Fixing leaky RyR2 may improve cardiac, pulmonary, and cognitive function in COVID‐19.

Article Snippet: Immunoblots were developed using the following primary antibodies: anti‐RyR2 (Affinity BioReagents, 1:2500), anti‐phospho‐RyR‐Ser(pS)‐2808 (Affinity BioReagents 1:1000), anti‐ calstabin2 (FKBP12 C‐19, Santa Cruz Biotechnology, Inc., 1:2500), and anti‐NOX2 (Abcam, 1:1000).

Techniques: Infection, Activation Assay, Protein-Protein interactions, Expressing, Activity Assay, Binding Assay

Journal:

Article Title: Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency

doi: 10.1016/j.jvc.2008.04.003

Figure Lengend Snippet: Polymerase chain reaction amplification primers use to evaluate the promoter and exons 1–4 of the canine calstabin2 gene

Article Snippet: Transcriptional activity of calstabin2 (Affymetrix ID, 1588953) was significantly different between the four groups of dogs ( P < 0.0002) ( ).

Techniques: Polymerase Chain Reaction, Amplification

Journal:

Article Title: Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency

doi: 10.1016/j.jvc.2008.04.003

Figure Lengend Snippet: Affected Boxer dogs display decreased transcriptional activity of calstabin2 as compared to control, Doberman pinschers with dilated cardiomyopathy, and dogs undergoing rapid ventricular pacing. Bar graph displaying relative transcriptional activity of calstabin2 in control, Doberman pinschers with dilated cardiomyopathy (DCM), dogs undergoing rapid ventricular pacing (Pacing), and Boxer dogs with arrhythmogenic right ventricular cardiomyopathy (Boxer) as measured by oligonucleotide microarray. *, P < 0.05 vs. control; †, P < 0.01 vs control; ‡, P < 0.001 vs. control; §, P < 0.05 vs. DCM.

Article Snippet: Transcriptional activity of calstabin2 (Affymetrix ID, 1588953) was significantly different between the four groups of dogs ( P < 0.0002) ( ).

Techniques: Activity Assay, Microarray

Journal:

Article Title: Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency

doi: 10.1016/j.jvc.2008.04.003

Figure Lengend Snippet: Boxer dogs with arrhythmogenic cardiomyopathy show decreased calstabin2 levels in the RyR2 channel complex. Cardiac lysates and SR microsomes were prepared from control animals and Boxer dogs with arrhythmogenic right ventricular cardiomyopathy (ARVC) as described in Section 2. (A) RyR2 was immunoprecipitated followed by immunoblotting as indicated. Calstabin2 is apparently decreased in the RyR2 complex while PKA phosphorylation at Ser2808 is unchanged. (B) Bar graph showing that calstabin2 normalized to cardiac RyR2 is significantly decreased in the channel complexes of Boxer dogs. *, P < 0.0001. (C) Immunoblot from whole cardiac tissue lysates (200 μg) separated by 15% PAGE developed with anti-calstabin antibody. Both calstabin1 (lower band corresponding to 12 kDa, indicated by dotted line and arrow) and calstabin2 (upper band corresponding to 12.6 kDa, indicated by solid line and arrow) are detected by the antibody in control dogs. Boxer dogs show reduced calstabin2 expression. Calstabin1 is present in cardiac tissues but has a much lower affinity for RYR2 than calstabin2. (D) RyR2 and calnexin protein levels from SR microsomes of control and Boxer dogs. (E) Quantification of RyR2 and calnexin is displayed in the left and middle panel, and RyR2 normalized to calnexin protein levels is shown in the right panel. No significant changes were detected (P values as indicated).

Article Snippet: Transcriptional activity of calstabin2 (Affymetrix ID, 1588953) was significantly different between the four groups of dogs ( P < 0.0002) ( ).

Techniques: Immunoprecipitation, Western Blot, Expressing