Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 interacts CaMKIIδ directly and promotes CaMKIIδ activation. A-B MS/MS spectrum of the peptide showing FTDEYQLFEELGK and ESTESSNTTIEDEDVK from CaMKIIδ. Single-letter abbreviations: K, Lys; V, Val; I, Ile; G, Gly; Q, Gln; L, Leu; N, Asn; E, Glu; T, Thr; S, Ser; F, Phe; D, Asp; Y, Tyr. m/z, mass/charge ratio. C The complex of JOSD2 and CaMKIIδ in NRVMs were detected by Coimmunoprecipitation. NRVMs were transfected with Flag-tagged JOSD2. Lysates from cells were immunoprecipitated with an anti-Flag antibody. CaMKIIδ levels were detected by immunoblotting. IgG was used as a control for IP. D In HEK-293 T cells which express a few endogenous CaMKII, Flag-tagged JOSD2 and GFP-tagged CaMKIIδ were transfected. Lysates from cells were immunoprecipitated with anti-Flag antibody. GFP levels were detected by immunoblotting. IgG was used as a control for IP. E Representative western blot for CaMKIIδ and p-CaMKIIδ in NRVMs with gradient levels of JOSD2. F–G JOSD2 was silenced or overexpressed in NRVMs. Representative western blotting for CaMKIIδ and p-CaMKIIδ in NRVMs. GAPDH was used as a loading control. (H-I) Representative western blotting for CaMKIIδ and p-CaMKIIδ in heart tissues from ISO- and MI-induced mice. J–K Confocal images (J)and representative recordings (K) of SR Ca2+ determined by the Fluo-4 AM fluorescence signal in cardiomyocytes transfected with si-JOSD2 or si-NC before ISO stimulation for 24 h. n = 3 cells in each group. [Scale bar, 50 μm]. L–M Confocal images (L) and representative recordings (M) of SR Ca2+ determined by the Fluo-4 AM fluorescence signal in cardiomyocytes transfected with Flag-JOSD2 or empty vector (EV) before ISO stimulation for 24 h. n = 6 cells in each group. [Scale bar, 50 μm]

Article Snippet: Antibodies against Calcium–calmodulin-dependent protein kinase II delta-Specific (CaMKIIδ; Cat# 20667-1-AP), Flag tag (Flag; Cat# 20543-1-AP), GFP tag (GFP; Cat# 66002-1-AP), beta-Myosin heavy chain (β-MyHC; Cat# 22280-1-AP), Collagen type I (COL-1; Cat# 14695-1-AP), transforming growth factor-beta1 (TGFβ-1; Cat# 21898-1-AP), and atrial natriuretic peptide (ANP; Cat# 27426-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Activation Assay, Tandem Mass Spectroscopy, Transfection, Immunoprecipitation, Western Blot, Control, Fluorescence, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 directly removes ubiquitin from CaMKIIδ at residue K227. A HEK-293 T cells were transfected with GFP-CaMKIIδ, Flag-JOSD2, Myc-Ub (WT), Myc-Ub(K48) and Myc-Ub(K63). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting. B A schematic illustration of the construct with mutated ubiquitination site of CaMKIIδ. (C) HEK-293 T cells were transfected with Flag-JOSD2, Myc-Ub, GFP-CaMKIIδ (WT), GFP-CaMKIIδK(K138R), GFP-CaMKIIδ(K227R) and GFP-CaMKIIδ(K268R). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting. D A schematic illustration of the construct with the mutated active site of JOSD2. E HEK-293 T cells were transfected with GFP-CaMKIIδ, Myc-Ub, Flag-JOSD2(WT) and Flag-JOSD2(C24A). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting

Article Snippet: Antibodies against Calcium–calmodulin-dependent protein kinase II delta-Specific (CaMKIIδ; Cat# 20667-1-AP), Flag tag (Flag; Cat# 20543-1-AP), GFP tag (GFP; Cat# 66002-1-AP), beta-Myosin heavy chain (β-MyHC; Cat# 22280-1-AP), Collagen type I (COL-1; Cat# 14695-1-AP), transforming growth factor-beta1 (TGFβ-1; Cat# 21898-1-AP), and atrial natriuretic peptide (ANP; Cat# 27426-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Ubiquitin Proteomics, Residue, Transfection, Western Blot, Construct