Journal: The Journal of General Physiology

Article Title: Obscurin deficiency leads to compensated dilated cardiomyopathy and increased arrhythmias

doi: 10.1085/jgp.202413696

Figure Lengend Snippet: Ca 2+ recirculation fraction and SERCA/PLB expression. (A) Representative Ca 2+ recirculation fraction estimated by the decline of potentiated beats following a period of high stimulation rate in trabeculae from trabeculae of 8-mo-old WT and Obscn KO male mice, stimulated at 5 Hz, in the absence and in the presence of Iso 0.1 µm. (B) Representative western blots for total SERCA, PLB, phospho-PLB at serine 16, and GAPDH. (C) Protein expression levels of WT ( n = 5) and Obscn KO ( n = 5) hearts excised from 8-mo-old male mice. The intensity of individual bands was quantified following normalization to that of GAPDH. The mean relative intensity of WT samples was set at 1. **P < 0.01 and ***P < 0.001 versus WT, as determined by Student’s t test. Source data are available for this figure: .

Article Snippet: Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) (Cell Signaling Technology) or 5% BSA in TBS-T for 1 h at room temperature, and then incubated overnight at 4°C with the following primary antibodies: anti-SERCA TRY2 used at 1:1,500 , anti-phospho-PLB (Ser16) used at 1:1,000 (cat. 07-052; Merck KGaA), anti-PLB used at 1:1,000 (cat. PA5-26004; Thermo Fisher Scientific), anti-GAPDH used at 1:2,000 (MA5-15738; Thermo Fisher Scientific) diluted in 5% nonfat dry milk or 5% BSA in TBS-T. Blots were washed with TBS-T and then incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (anti-rabbit IgG, HRP-linked antibody, cat. 7074, anti-mouse IgG, HRP-linked antibody, cat. 7076; Cell Signaling Technology) diluted 1:2,000 or 1:3,000 in 5% BSA or 5% milk in TBS-T solution.

Techniques: Expressing, Western Blot