Journal: Neurobiology of disease

Article Title: Ethanol-induced ceramide production causes neuronal apoptosis by increasing MCL-1S-mediated ER-mitochondria contacts.

doi: 10.1016/j.nbd.2023.106009

Figure Lengend Snippet: Fig. 4. IP3R-MCL-1S interaction promotes F-actin polymerization through CaM-mediated INF2 activation. A SH-SY5Y cells were incubated with EtOH for 24 h. ER fraction was mentioned in methods. MCL-1S was detected by western blot. Anti-Calnexin was used as ER marker. n = 5. B, C SH-SY5Y cells and iPSC-neurons were treated with EtOH for 24 h. IP3R was immunoprecipitated with anti-MCL-1S and anti-IP3R antibodies (left). The expression of MCL-1S, IP3R, and β-Actin in total cell lysates is shown (right). n = 5. D SH-SY5Y cells treated with EtOH for various time and then loaded with Mag-Fluo-4 (1 μM). The amount of ER calcium was measured by using flow cytometer. n = 5. E SH-SY5Y cells were transfected with MCL-1S siRNA or NT siRNA for 24h prior to EtOH exposure for 24 h. Mag-Fluo-4-positive cells were analyzed with flow cytometer. n = 5. F SH-SY5Y cells were immunostained with CaM antibody and ER. ER was stained by ER dye (red). Co-localization of CaM (green) and ER (red) was visualized with SRRF imaging system. Scale bars are 8μm. n = 5. G SH-SY5Y cells were transfected with INF2-CAAX siRNA or NT siRNA for 24h prior to EtOH exposure for 24 h. Mitochondrial fraction was mentioned in methods. F-actin was detected by western blot. Anti-TOMM20 was used as mitochondria marker. n = 5. H SH-SY5Y cells were immunostained with phalloidin and TOMM20 antibody. Co- localization of phalloidin (green) and TOMM20 (red) was visualized with SRRF imaging system. Scale bars are 5μm. n = 5. All blot, immunofluorescence, and flow cytometer images are representative. Quantitative data are shown as a mean ± S.E.M. * indicates p < 0.05 vs. control, # indicates p < 0.05 vs. EtOH. (For inter pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: INF2 antibody (NBP1–79025) was purchased from Novus Biologicals.

Techniques: Activation Assay, Incubation, Western Blot, Marker, Immunoprecipitation, Expressing, Flow Cytometry, Transfection, Staining, Imaging, Immunofluorescence, Control

Journal: Nature Cardiovascular Research

Article Title: Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram

doi: 10.1038/s44161-023-00294-y

Figure Lengend Snippet: Immunoprecipitated channels Scn5a a ), Kcnq1 b ), and Cacna1c c ) evaluated by western blot. d ) Inf2 (Cacna1c interactor) was immunoprecipitated from murine cardiac tissue and evaluated by western blotting for the presence of Cacna1c (left panel, probed with IRDye 800CW secondary antibody) to confirm co-immunoprecipitation with Inf2. The same blot was evaluated for presence of Inf2 (right panel, probed with IRDye 680LT secondary antibody) to ensure that Inf2 was immunoprecipitated. In all the panels, black arrows denote the band of interest. The lowermost arrow in Panel B shows the Kcnq1 monomer band and the higher order oligomers are shown by the upper arrows. UF denotes unbound fraction of the immunoprecipitation (IP) experiment. The immunoblot validation was carried out thrice for panels A-C and twice for panel D with reproducible results. e ) LC-MS/MS analysis of Inf2 IPs evaluating Cacna1c (left) and Inf2 protein abundances (right). Triplicate immunoprecipitations were performed from murine cardiac tissue using antibodies against Inf2 or IgG. Precipitated proteins were evaluated by mass spectrometry. Inf2 was abundantly present in the three Inf2 IPs and was absent in the triplicate IgG control Ips (right). The calcium channel protein Cacna1c was identified in all six immunoprecipitations but was a hundred-fold more abundant in the Inf2 IPs than in the control IPs (left).

Article Snippet: IP was carried out using Anti-Inf2 antibody (Bethyl Labs, A303-428A), and the whole eluate was loaded on SDS–PAGE gels.

Techniques: Immunoprecipitation, Western Blot, Biomarker Discovery, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Control

Journal: Nature Cardiovascular Research

Article Title: Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram

doi: 10.1038/s44161-023-00294-y

Figure Lengend Snippet: The functional consequences after acute KO of six interactors of three major ion channels—Kcnq1 (Nebl and Nrap), Cacna1c (Inf2) and Scn5a (Glipr2, Epn2 and Gsn)—were evaluated in zebrafish. a , Compared with control siblings (WT, n = 16 fish), KO of the gene encoding Kcnq1-interacting nebl (nebl KO, n = 23) led to prolongation of the ventricular action potential starting shortly after the plateau, with the greatest effect size at slower heart rate and later in repolarization (APD 80 , action potential duration measured at 80% recovery; bpm, beats per minute; ** P = 8.4 × 10 −3 ; * P = 0.037 by two-sided Mann–Whitney U test; exemplar amplitude-normalized optical action potentials shown below), reduced ventricular CV (sinus rhythm; ** P = 4.2 × 10 −3 by idem; exemplar relative activation time maps are zero-referenced to activation of the AV-Ring; white stars indicate area of global earliest activation, isochrones denote 5 ms intervals), and increased spatial dispersion of repolarization (σ-Repol 80 , standard deviation of repolarization time at 80% recovery across the chamber; sinus rhythm; ** P = 8.74 × 10 −3 , idem; in exemplar relative repolarization 80 time maps, each chamber is zero-referenced to median repolarization time). b , KO of Kcnq1-interacting nrap ( n = 29) led to prolongation of ventricular action potential with greatest effect size in early repolarization (APD 20 ) and at faster heart rates (* P = 0.016; ** P = 5.22 × 10 −3 , idem, n WT = 27). c , Knockdown of Cacna1c-interacting inf2 ( n = 10) caused a significant decrease in ventricular CV (*** P = 7.2 × 10 −4 , idem, n WT = 7). d , KO of Scn5a-interacting glipr2/glipr2l ( n = 14) decreased ventricular CV (* P = 0.013) and rate of the action potential upstroke ( V max ; *** P = 2.61 × 10 −4 , exemplar amplitude normalized action potential upstrokes shown) as well as increasing ventricular APD (** P = 2.7 × 10 −3 , all by idem, n WT = 9). e , KO of Scn5a-interacting epn2 ( n = 16) resulted in decreased ventricular CV (* P = 0.020) and increased ventricular APD 80 (* P = 0.035, idem, n WT = 18). f , Decrease in ventricular CV was also observed after KO of Scn5a-interacting gsna/b ( n = 11, * P = 0.027, idem, n WT = 10). Each point in the box plots corresponds to an individual zebrafish embryo. Box plots indicate 25th/50th/75th percentiles, while whiskers extend to the most extreme data within 1.5× of interquartile range beyond box limits.

Article Snippet: IP was carried out using Anti-Inf2 antibody (Bethyl Labs, A303-428A), and the whole eluate was loaded on SDS–PAGE gels.

Techniques: Functional Assay, Control, MANN-WHITNEY, Activation Assay, Dispersion, Standard Deviation, Knockdown

Journal: Nature Cardiovascular Research

Article Title: Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram

doi: 10.1038/s44161-023-00294-y

Figure Lengend Snippet: Signal-average trace (black) overlaid R-aligned traces from individual beats (green). a ) Nrap knockout ECGs displayed repolarization abnormalities such that T-waves were not discernible from signal noise (red underscored period). b ) ECGs from inf2 knockout fish had continued rising activity throughout the PR segment without achieving an isoelectric level as typically seen in wildtype or other knockouts (red underscored period). c ) ECGs from pde4dip knockout fish displayed greater variance in signal waveform between individual beats showing substantial deviations from mean well in excess of typical noise (extrema envelope in red).

Article Snippet: IP was carried out using Anti-Inf2 antibody (Bethyl Labs, A303-428A), and the whole eluate was loaded on SDS–PAGE gels.

Techniques: Knock-Out, Activity Assay