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: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 1. Kaplan–Meier overall survival and correlations between ERBB2 and formin mRNA expression. (A) Kaplan–Meier OS analyses of HER2-positive breast cancer samples reveal significantly poorer prognosis associated with FHOD1, INF2, and DAAM1 mRNA expression. The number of cases is denoted at the bottom of the figures. (B) ERBB2 mRNA expression in HER2-positive subtype breast tumors (n = 230) exhibits a significant positive correlation with FHOD1, INF2, and DAAM1. Kaplan–Meier analyses and correlations for all formins, excluding GRIDIP, are depicted in Supplementary Figures 1 and 2. ERBB2 = erb-b2 receptor tyrosine kinase 2; HER2 = human epidermal growth factor receptor 2; OS = overall survival; HR = hazard ratio.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Expressing

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 2. Immunohistochemistry. Tissue microarrays were sectioned at 3.5 μm and immunostained using FHOD1, INF2, or DAAM1 antibodies, following the streptavidin-peroxidase technique. The samples were evaluated based on the predominant staining intensity and categorized into four levels: negative, low, moderate, or strong. No instances of strong INF2 intensity were detected. Magnification: 400×. Scale bars = 50 µm.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Immunohistochemistry, Staining

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 3. Localization of FHOD1, INF2, and DAAM1 in MDA-MB-453 and SK-BR-3 breast cancer cell lines. Upper panel: FHOD1 (red) is predominantly seen as cytoplasmic dots. Middle panel: INF2 (red) is distributed in the cytoplasm colocalized with phalloidin (green) and lamellipodia. Lower panel: DAAM1 (red) appears as cytoplasmic dots with co-localization with actin filaments. Magnification: 400×. Scale bars = 20 µm.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques:

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 4. Small interfering RNA-mediated knockdown of FHOD1, INF2, DAAM1, or their combinations. (A) Knockdown effectiveness was assessed through immunoblotting 48-hour post-transfection, with GAPDH as a loading control. (B) Representative images of transfected cells with control, FHOD1, INF2, DAAM1, or combined siRNAs. Alexa Fluor 488-conjugated phalloidin and DAPI were used to visualize actin filaments and nuclei, respectively. Magnification: 400×. Scale bars = 50 µm. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; siRNA = small interfering RNA; DAPI = 4′,6-diamidino-2-phenylindole.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Small Interfering RNA, Knockdown, Western Blot, Transfection, Control

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 5. Effect of knockdown of FHOD1, INF2, DAAM1 or their combination in wound healing, transwell migration, and proliferation. (A) Scratch wounds were performed 48-hour post siRNA transfection, and the medium subsequently replaced by starvation medium or starvation medium supplemented with 50 ng/mL HRG to speed migration. The wound relative densities were monitored over 96 hours. (B) Forty-eight hours after transfection, cells were seeded on the upper surface of the transwell migration chamber, while the lower chamber contained medium supplemented with 10% serum as a chemoattractant. Confluence of cells on the bottom surface of the membrane was monitored for 96 h and normalized based on the initial top surface cell area for each well. (C) For proliferation assessment, cells were plated to cover 5%–10% of the area 48-hour post-transfection, and the growth of confluence measured over 144 hours. Comparisons between cells treated with control siRNA and those subjected to FHOD1, INF2, DAAM1, and their combinations were analyzed using Student’s t-test, where *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 were considered statistically significant. siRNA = small interfering RNA; HRG = heregulin.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Knockdown, Migration, Transfection, Membrane, Control, Small Interfering RNA

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 6. HER2/ERBB2 knockdown alters FHOD1 and INF2 expression through Akt and MAPK pathways. (A) Subcellular localization of HER2 (in red) in MDA- MB-453 and SK-BR-3 cells, with ERBB2 concentrated in actin-rich cellular protrusions, observed through phalloidin staining (green). DAPI (blue) indicates nuclei. Magnification: 400×. Scale bars = 20 µm. (B) Cells were treated with ERBB2 siRNA for 48 hours, and the knockdown efficacy validated by western blotting in both MDA-MB-453 and SK-BR-3 cells. In MDA-MB-453 cells, HER2/ERBB2 knockdown decreased FHOD1, INF2, and DAAM1 levels, concomitant with a reduction in pMAPK. In SK-BR-3 cells, ERBB2 knockdown reduced FHOD1 and INF2 expression, accompanied by lowered Akt/pAkt and MAPK phosphorylation levels. Both cell lines were treated with MEK1/2 inhibitor UO126, PI3K inhibitor LY294002, or the control vehicle DMSO. Effective pathway inhibition was verified by western blotting of Akt and MAPK phosphorylation states. Treatment with PI3K or MAPK inhibitors in MDA-MB-453 cells emulated the effects of HER2/ERBB2 knockdown, decreasing FHOD1 and INF2 expression. In contrast, inhibiting PI3K or MAPK alone did not induce alterations in FHOD1 and INF2 levels in SK-BR-3 cells. Notably, DAAM1 levels remained unaffected by PI3K or MAPK inhibitor treatments in both cell lines. HER2 = human epidermal growth factor receptor 2; ERBB2 = erb-b2 receptor tyrosine kinase 2; DAPI = 4′,6-diamidino-2-phenylindole; siRNA = small interfering RNA.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Knockdown, Expressing, Staining, Western Blot, Phospho-proteomics, Control, Inhibition, Small Interfering RNA

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: The Journal of Physiological Sciences : JPS

Article Title: Long-term resistance exercise-induced muscular hypertrophy is associated with autophagy modulation in rats

doi: 10.1007/s12576-017-0531-2

Figure Lengend Snippet: Changes in autophagy flux-related proteins (LC3 and p62) and autophagy induction-related proteins (BECLIN1, BNIP3, and ATG7) in FDP muscles after 8 weeks of resistance exercise training. a Representative Western-blot images for LC3 and p62 in each group. b Quantification of LC3-I, LC3-II, and LC3-II/I ratio. c Quantification of p62. d Representative Western-blot images for BECLIN1, BNIP3, and ATG7 in each group. e Quantification of BECLIN1. f Quantification of BNIP3. g Quantification of ATG7. Comparisons of each target protein expression were made after normalization to GAPDH. SC sedentary control group, RE resistance exercise group. *p < 0.05 vs. SC

Article Snippet: The antibodies used are as follows: AKT (9272, 1:1000), p-AKT Ser473 (9271, 1:1000), mTOR (2972, 1:1000), p-mTOR Ser2448 (2971, 1:1000), AMPKα (2532, 1:1000), p-AMPKα Thr172 (2535, 1:1000), ULK1 (8054, 1:1000), p-ULK1 Ser757 (14202, 1:1000), LC3 (4108, 1:1000), p62 (5114, 1:1000), BECLIN1 (3738, 1:1000), and ATG7 (2631, 1:1000) from Cell Signaling Technology; BNIP3 (10433, 1:500), TFEB (2636, 1:500), LAMP-2 (13524, 1:500), and CATHEPSIN L (133641, 1:500) from Abcam; p-ULK Ser555 (ABC124, 1:2500) from Millipore; p70S6K (SC-8418, 1:200), p-p70S6KT Ser411 (SC-8416, 1:200) from Santa Cruz Biotechnology.

Techniques: Muscles, Western Blot, Expressing, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: Long-term resistance exercise-induced muscular hypertrophy is associated with autophagy modulation in rats

doi: 10.1007/s12576-017-0531-2

Figure Lengend Snippet: Summary of RE-induced muscular adaptation. RE-induced increase in muscle fiber cross-sectional area and strength is caused by modulating the rate of protein turnover, with protein synthesis predominating protein degradation. RE enhances an anabolic signaling nexus of hypertrophy pathways (AKT-mTOR-p70s6k) but suppresses a catabolic process, autophagy, which was evidenced by reduced AMPK phosphorylation levels, a declined LC3-I/LC3-II ratio, diminished active CATHEPSIN levels, and increased p62 levels. Our data suggest that although showing only a limited snapshot of metabolic change 24 h after the last bout of RE, sustained anabolic signaling activation may restrict autophagy-related catabolic processes, contributing to an increase in muscle fiber sizes

Article Snippet: The antibodies used are as follows: AKT (9272, 1:1000), p-AKT Ser473 (9271, 1:1000), mTOR (2972, 1:1000), p-mTOR Ser2448 (2971, 1:1000), AMPKα (2532, 1:1000), p-AMPKα Thr172 (2535, 1:1000), ULK1 (8054, 1:1000), p-ULK1 Ser757 (14202, 1:1000), LC3 (4108, 1:1000), p62 (5114, 1:1000), BECLIN1 (3738, 1:1000), and ATG7 (2631, 1:1000) from Cell Signaling Technology; BNIP3 (10433, 1:500), TFEB (2636, 1:500), LAMP-2 (13524, 1:500), and CATHEPSIN L (133641, 1:500) from Abcam; p-ULK Ser555 (ABC124, 1:2500) from Millipore; p70S6K (SC-8418, 1:200), p-p70S6KT Ser411 (SC-8416, 1:200) from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Activation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: The N-terminal extension of INF2 contains two α-helices. A INF2 has an extension N-terminal to the DID that is shorter than that of mDia1 and lacks a G domain. G (also known as GBD N ) domain necessary for Rho binding that is adjacent to the N terminus of the DID, GBD GTPase-binding site, DAD diaphanous autoinhibitory domain, DID diaphanous inhibitory domain, FH1 and FH2 formin homology 1 and 2, respectively. B Ribbon representation of the NMR solution structure of the N-terminal extension of INF2. Backbone atoms are shown in black. K and R side chains are displayed in blue, D and E side chains in red, and all others in green

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: The first of α-helix of INF2 is required for the normal distribution and content of F-actin. A Schematic of the INF2 proteins expressed. B–D INF2 KO cells or INF2 KO cells expressing mCherry alone or mCherry fusions of the indicated INF2 proteins were stained for F-actin. Nuclei were visualized with DAPI. An enlargement of the DAPI and F-actin staining corresponding to the boxed regions is shown in the rightmost panels. Scale bar, 10 μm ( B ). Box plots showing the intensity of F-actin staining at the perinuclear region ( C ) and the cytosol ( D ) relative to that of control INF2 KO cells (> 140 cells were analyzed for each experimental condition; three independent experiments; ns not significant; *** p < 0.001)

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Expressing, Staining, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: CaM binds the N-terminal α-helix of INF2. A Schematic of the INF2 fragments used in the pull-down experiments. B , C Pull-down analysis of the association of GFP-CaM with GST fusions of the indicated INF2 fragments. D GST fusions of the fragment comprising amino acids 4–19 of INF2 with the indicated mutations in residues W11, L14 and L18 were used in pull-down experiments with GFP-CaM. The purified GST fusions were stained with Coomassie blue as a control for the amount of GST fusions used in ( B – D ). E Lysates of HEK293T cells expressing intact INF2 or INF2 with the triple W11L14L18A mutation fused to mCherry were immunoprecipitated with control or anti-Cherry antibodies and immunoblotted for exogenous INF2 with anti-Cherry antibodies, and with antibodies to endogenous CaM

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Purification, Staining, Control, Expressing, Mutagenesis, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: Surface plasmon resonance analysis of the interaction of the 2-19 INF2 peptide with CaM. A – D Representative equilibrium binding affinity measurements of the interaction of purified CaM from bovine brain with the control 2-19 peptide (A, left panel) or 2-19 peptides containing the L14L18A (B), W11A (C) and W11L14L18A (D) mutations in the presence of Ca 2+ . E Control of the binding of CaM to the 2-19 peptide in the absence of Ca 2+ . The equilibrium plots of CaM with the control 2-19 (A, right panel) is shown, with indication of the apparent K D resulting from the same experiment. RU resonance units, Req RU at equilibrium

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: SPR Assay, Binding Assay, Purification, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: Conservation of the CaMBS of INF2. A Alignment of the sequence of the 1–19 peptide of human INF2 with that of 50 species from different groups of vertebrates. The corresponding position of the W11, L14 and L18 residues of human INF2 is indicated. B Compilation of allelic variants of the 6–19 sequence of INF2 in the general human population, with indication of the SNP reference, database and frequency. C Examples of CaM-binding proteins with a sequence similar to the 1-4-8 motif of INF2. AKAP79 A-kinase-anchoring protein 79, CX32 connexin 32, GRM5 glutamate metabotropic receptor 5, PDE1A and PDE1B Ca 2+ /CaM-dependent 3´,5´-cyclic nucleotide phosphodiesterases 1A and 1B, RIT1 and RIT2 Ras-related GTPases 1 and 2, TRPV4 transient receptor potential vanilloid subtype 4

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Sequencing, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: Structural model of the CaM/CaMBS complex. A Structural model of C-lobe of CaM bound to the 2-19 INF2 peptide. The position of the three hydrophobic amino acids comprising the 1-4-8 motif of the CaMBS of INF2 is indicated. B GST, alone or fused to the 2–21 peptide of INF2, was used in pull-down experiments with GFP fused to intact (WT) or to CaM with mutations that inactivate the two lobes (mutant 1234), the N-terminal lobe (mutant 12) or the C-terminal lobe (mutant 34). The purified GST fusions were stained with Coomassie blue as a control for the amount of GST protein used

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Mutagenesis, Purification, Staining, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: Integrity of the CaMBS of INF2 is crucial for actin homeostasis. A – C INF2 KO cells expressing mCherry fusions of the indicated INF2 mutants were stained for F-actin. Nuclei were visualized with DAPI. An enlargement of the DAPI and F-actin staining corresponding to the boxed region is shown in the rightmost panels. Scale bar, 10 μm(A). Box plots showing the intensity of F-actin staining at the perinuclear region ( B ) and the cytosol ( C ) relative to that of control INF2 KO cells (> 150 cells were analyzed for each experimental condition; three independent experiments; ns not significant; * p > 0.05; *** p < 0.001)

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Expressing, Staining, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: The CaMBS of INF2 is required for actin polymerization and incorporation of GFP-MRTF-A into the nucleus in response to increased intracellular Ca 2+ levels. A , B GCaMP6S INF2 KO cells expressing the indicated exogenous INF2 proteins were incubated with SiR-actin to visualize F-actin. Cells were treated with A23187 (0 min) and analyzed by videomicroscopy before and after treatment ( A ). Note that, although the staining of F-actin with SiR-Actin in live cells was poor compared to that of fluorescent phalloidin in fixed cells, SiR-Actin was useful for detecting the initial increase of the levels of F-actin after A23187 stimulation. B Kinetics of the perinuclear content of F-actin as measured by SiR-actin fluorescence relative to that of cells at t = 0 min (> 30 cells were analyzed for each experimental condition; three independent experiments). C – E GFP-MRTF-A INF2 KO cells expressing mCherry fusions of intact INF2 or the INF2 W11L14L18A mutant were treated with A23187 and analyzed by videomicroscopy before and after treatment ( C ). ( D ) Kinetics of the number of INF2 KO cells with nuclear distribution of GFP-MRTF-A relative to that of cells at t = 0 min (≥ 30 cells were analyzed for each experimental condition; three independent experiments). Scale bars, 5 μm. (E) The histogram shows the percentage of cells with GFP-MRTF-A predominantly in the nucleus after 5 min of treatment (> 190 cells were analyzed for each experimental condition; 3 independent experiments; *** p < 0.001). The mean ± SD is shown in ( B , D , E )

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Expressing, Incubation, Staining, Fluorescence, Mutagenesis

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet: Comparative schematics of the activation of mDia1 and INF2. mDia1 is activated by the binding of GTP-loaded Rho to the GBD, whereas INF2 activation takes place when Ca 2+ /CaM binds to the INF2 N-terminal extension

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Activation Assay, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet:

Article Snippet: Peptide INF2 2-36 c KGGSVKEGAQRKWAALKEKLGPQDSDPTEANLESADPE , pepMic Co., Ltd , N/A.

Techniques: Control, Protease Inhibitor, Mutagenesis, DNA Purification, Recombinant, Software