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

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 1: INF2 splice variants. (A) Domain architecture of INF2. C-Terminal sequences starting at K1224 of human INF2. The CAAX and non-CAAX splice variants are shown in purple and green amino acids, respectively. (B) Schematic of posttranslational modifications associated with prenylation.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 2: INF2 does not localize to ER in U2OS cells. (A) U2OS cells transfected with mCherry- Sec61β (an ER membrane protein; red) and stained with anti-INF2 antibody (green) and DAPI (blue). (B) NIH 3T3 cells stained with anti-INF2 (green), anti-GRP94 (an ER luminal protein; red) and DAPI (blue). See Supplemental Figure S1B for analogous INF2/ER localization using the ER marker. Scale bar, 20 μm.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Transfection, Membrane, Staining, Marker

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 3: Actin depolymerization causes INF2 aggregation in U2OS cells. (A) U2OS cells were treated with 1 μM LatB for 0 and 30 min prior to fixation and staining with anti-INF2 (green), TRITC-phalloidin (red), and DAPI (blue). Arrow indicates one such actin aggregate where INF2 colocalizes. Scale bar, 20 μm. (B) Single confocal slice of U2OS cells stained with anti-INF2 (green) and rhodamine-phalloidin (red). Arrows show examples of regions where INF2 and actin filaments colocalize. Scale bars: 5 μm (leftmost three images) and 20 μm (rightmost image).

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Staining

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 4: 3T3 cells express INF2-CAAX, whereas U2OS, Jurkat, and HeLa cells express INF2–non-CAAX. (A) Western blots of whole-cell homogenates from NIH 3T3 cells. CAAX and non-CAAX controls are from U2OS cells transiently transfected with GFP-tagged mouse INF2 isoforms. Western blots were probed with antibodies specific for total mouse INF2, INF2-CAAX, or INF2–non-CAAX. (B) Similar analysis as in A, using homogenates from Jurkat and HeLa cells. CAAX and non-CAAX controls are from U2OS cells transiently transfected with GFP-tagged human INF2 isoforms. (C) Western blots of immunoprecipitations of denatured lysates from U2OS cells either untransfected or stably expressing GFP-INF2-CAAX or GFP-INF2–non-CAAX. Immunoprecipitations were conducted using the total INF2 antibody. Western blots were probed with total INF2, INF2-CAAX, or INF2–non-CAAX antibodies. E, endogenous INF2; G, GFP INF2.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Western Blot, Transfection, Stable Transfection, Expressing

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 5: INF2 is rapidly extracted from U2OS cells. U2OS cells were permeabilized with digitonin on ice for 0, 5, and 30 min before fixation. The cells were then stained with anti-INF2 antibody (green) to detect endogenous INF2 and DAPI (blue). mCherry-Sec61β (red) was used as an ER membrane marker. Scale bar, 20 μm.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Staining, Membrane, Marker

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 6: Stably transfected U2OS cells lines expressing GFP-INF2-CAAX and GFP-INF2–non- CAAX recapitulate endogenous proteins. (A) Localization of GFP-INF2-CAAX and GFP-INF2– non-CAAX in U2OS cells. mCherry-Sec61β (red) was used as an ER marker. Scale bar, 10 μm.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Stable Transfection, Transfection, Expressing, Marker

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 7: INF2 enriches around the Golgi apparatus. (A) U2OS cells were stained with anti-INF2 (green) and golgin-97 (a marker for the trans-Golgi network; red). Top, one confocal z-section. Bottom, the maximum-intensity projection of multiple confocal z-sections. Arrow represents INF2 enrichment around the Golgi. Scale bar, 20 μm. (B) U2OS cells treated for 0 or 30 min with 2.5 μg/ml brefeldin A. Arrow represents INF2 enrichment around the Golgi The cells were then fixed and stained with anti-INF2 (green) and anti–golgin-97 (red) antibodies. Scale bar, 20 μm.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Staining, Marker

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 8: INF2 depletion causes dispersal of the Golgi apparatus in U2OS cells. (A) INF2- depleted U2OS cells stained with anti-INF2 (green) and DAPI (blue). The stars indicate cells that have been knocked down. Scale bar, 20 μm. (B) Western blot showing INF2 depletion using two different siRNAs. Tubulin was used as a loading control. (C) Representative images of compact, spread, and dispersed Golgi. GM130 (green) was used as the Golgi marker. DAPI is in blue. Scale bar, 20 μm. (D) Quantification of Golgi dispersion. The data represent the average of three independent experiments (n > 200 cells). Error bars, SE. (E) Quantification of Golgi morphology upon INF2 depletion and LatB treatment. C, control siRNA; S, INF2 siRNA. Cells were treated with 0, 1, or 2 μM LatB for 1 h prior to fixation.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Staining, Western Blot, Control, Marker, Dispersion

Journal: Molecular Biology of the Cell

Article Title: Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

doi: 10.1091/mbc.e11-05-0457

Figure Lengend Snippet: Figure 9: Peri-Golgi actin patches are reduced and dispersed in INF2 knockdown. (A) Cells transfected with control (top) or INF2 (bottom) siRNAs were fixed and stained with anti-GM130 (green) and TRITC-phalloidin (red). Maximum-intensity projections of three consecutive 0.2-μm sections taken through the middle of Golgi region are shown. Arrows indicate actin patches found in close proximity to Golgi stacks. Scale bar, 10 μm. Quantification of peri-Golgi actin patches reveals an approximate 30% decrease in patch number upon INF2 knockdown (12.7 patches/Golgi in control siRNA [217 patches in 17 cells] vs. 9.0 in INF2 siRNA [190 patches in 21 cells]). Quantification is the result of two separate experiments that showed similar 30% decrease for INF2 siRNA. (B) Colocalization of INF2 with actin patches. Single confocal slice of U2OS stained with anti-INF2 (green), TRITC-phalloidin (red), and anti–golgin-97 (blue). Arrow indicates a patch that colocalizes with INF2, and arrowhead indicates a patch that does not colocalize with INF2. Scale bar, 5 μm.

Article Snippet: Human INF2 ORF clone (SC313010) was obtained from OriGene (Rockville, MD).

Techniques: Knockdown, Transfection, Control, Staining

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