cx43 phosphorylated ser368 (pcx43  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative western blots suggest that neither total connexin43 (Cx43) nor Cx43 phosphorylated at serine-368 (pCx43-S368) significantly change in response to acute exposure to osmotic agents. Lane 7 contains a positive control using hearts exposed to 60 min of room temperature ischemia, a condition shown to upregulate Cx43 pS368. B. Summary data plotted with mean and standard deviation error bars do not reveal significant differences in either total or pCx43-S368 for all conditions (N=3 hearts per condition. p=n.s. via ordinary one-way ANOVA applying Dunnett’s correction). C. Representative fluorescent images of calcein-AM fluorescence in control cultures (N=13 plates, n=220 cells) and cultures exposed to mannitol (N=7 plates, n=116 cells), albumin (N=4 plates, n=77 cells), Dextran 70kDa (N=4 plates, n=102 cells), Dextran 2MDa (N=4 plates, n=95 cells), and carbenoxolone (CBX, N=6 plates, n=110 cells). Measurements were made before bleaching (Pre-bleach), just after bleaching (t=0s), halfway through and at the end of the recovery period (t=150 and 300s, respectively). D. Fluorescence recovery after photobleaching (FRAP) plot of bleached cells during 300s post bleaching. Solid lines represent averages at each point, and shaded areas represent the standard error of the mean. E. Normalized recovery demonstrates that only CBX significantly decreased FRAP relative to control. *p<0.05, nested ANOVA applying Dunnett’s correction with culture plate nested under treatment type.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Western Blot, Positive Control, Standard Deviation, Fluorescence

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative images of ventricular tissue slices perfused with 0.5, 3, and 10kDa fluorescent dyes. Panels demonstrate extracellular diffusion; Cx43 signal is used to identify the intercalated disc (ID), and nuclei are shown alone and with overlay of all signals. B. A region of interest is identified around the Cx43 ID signal and intensity of signal is averaged to identify the peak of the Cx43 signal. The same spatial averaging with 0.5, 3, and 10kDa signal demonstrates a dip in 3 and 10kDa signal with a peak in the 0.5kDa dye demonstrating the 0.5kDa dye permeates the ID, but the other two dyes do not. C. The intensity of the dye fluorescent signal at the peak of the Cx43 signal is normalized to the intensity of signal beyond the ID to reveal that 3 (n=3 images) and 10kDa (n=4 images) fluorescent signal are significantly lower than 0.5kDa (n=7 images) signal intensity. *p<0.05 relative to 0.5kDa, one way ANOVA with Dunnett’s correction.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Diffusion-based Assay