Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Schematic diagram of the INF2 domains, highlighting the regions targeted by polyclonal antibodies, the disease-associated residue R218, as well as the 1-420, ΔNT, and FFC constructs used in this study. Domain boundaries (human INF2): N-terminus, 1-34; DID, 35-259; FH1, 421-520; FH2, 535-941; DAD, 967-995. (B) Schematic of the cell-free assay. U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX are homogenized, followed by low speed centrifugation (600xg), after which the pellet (LSP) is used. Actin polymerization activity of the LSP is measured using pyrene actin assay, in the presence of actin monomer (2 μM, 5% pyrene-labeled), capping protein (50 nM) and profilin (6 μM). (C) U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX or GFP were treated with vehicle or 100 μM histamine for 30 seconds, then fixed and stained with TRITC-phalloidin (to visualize actin filaments) and DAPI (to stain the nucleus). Scale bar: 10 μm; boxed region: 12 × 12 μm. (D) Western blots of total cell lysate and LSP from either U2-OS INF2 KO cells or GFP-INF2-CAAX stable cells. Equal protein (5 μg) was loaded in each lane and blot was probed with the indicated antibodies. (E, F) Representative pyrene-actin polymerization assays using LSP from GFP-INF2-CAAX stable cells (E) or from INF2 KO cells (F). Each reaction contains 10 μg LSP protein, 2 μM added actin monomer (5% pyrene-labeled), 50 nM capping protein, and 6 μM profilin, with or without the indicated components (167 nM anti-DID antibody, 167 nM anti-FH1FH2 antibody, 1 μM calcium with 250 nM CALM, as specified). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Residue, Construct, Cell-Free Assay, Stable Transfection, Expressing, Centrifugation, Activity Assay, Pyrene Actin Assay, Labeling, Staining, Western Blot

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Pyrene actin polymerization assays containing 2 µM actin (5% pyrene-labeled), 10 nM INF2 full-length or 10 nM INF2-FFC protein. Experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (B) Extended western blots corresponding to . Equal total protein (5 μg) from total cell lysate or LSP of either U2-OS INF2 KO cells or GFP-INF2-CAAX stable cells was loaded and blotted with the indicated antibodies. (C) Left: Western blots detecting actin and GAPDH in LSP and total cell lysates from U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX. Right: Table showing the calculated amounts of actin in LSP and total cell lysates. See Materials and Methods for details. (D) LSPs prepared from either U2-OS INF2 KO cells or cells stably expressing GFP-INF2-CAAX were stained with TRITC-phalloidin and DAPI to visualize filamentous actin associated with LSPs. Scale bar: 10 μm. (E) Representative western blots showing actin in the supernatant (Sup) and pellet fractions of LSPs from the indicated cells, LSP treatment with either 2 μM phalloidin + 2 μM LatA, or 2 μM LatA alone overnight. (F) The percentage of actin in the supernatant and pellet fractions in LSPs prepared with or without phalloidin. Experiment was performed twice. (G) Extended data for figure 2E. Pyrene fluorescence intensity of actin polymerization reactions was measured following overnight incubation. Reactions were performed using 2 μM actin (5% pyrene-labeled), 50 nM capping protein, and 6 μM profilin, with or without 10 μg of LSP from U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX wild-type. Where indicated, 167 nM anti-DID or 167 nM anti-FH1FH2 antibody was included. (H) Pyrene-actin polymerization assay containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg LSP from GFP-INF2-CAAX stable cells, with or without 1 μM calcium, 100 nM CALM and 100 μM W-7 (calmodulin inhibitor). Experiments were performed twice with similar results, and the data shown are a representative experiment

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Labeling, Western Blot, Stable Transfection, Expressing, Staining, Fluorescence, Incubation, Polymerization Assay

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Pyrene-actin polymerization assay containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg LSP from GFP-INF2-CAAX stable cells, with or without 1 μM calcium and 1 μM CALM. Experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (B, C) Titration curves for CALM in the presence of 1 μM calcium (B) and free calcium in the presence of 1 μM CALM (C). Polymerization rates were calculated from the linear phase of the pyrene-actin assay (0-500 seconds). nH = Hill coesicient. (D) Pyrene-actin polymerization assay performed as in (A), with the indicated components (1 μM calcium, 250 nM CALM and/or 670 nM anti-FH1FH2 antibody). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (E, F) Representative confocal microscopy images showing polymerized actin around LSP from GFP-INF2-CAAX stable cells (E) or INF2 KO cells (F). Each reaction contains 10 μg LSP, 2 μM added actin monomer (10% TAMRA-labeled), 50 nM capping protein, and 6 μM profilin, with or without the indicated components (167 nM anti-DID antibody, 167 nM anti-FH1FH2 antibody, 1 μM calcium with 100 nM CALM, as specified).

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Polymerization Assay, Labeling, Titration, Pyrene Actin Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Time-lapse montage of U2-OS INF2 KO cells transiently expressing mApple-F-tractin along with either GFP-INF2 wild-type or GFP-INF2 R218Q, stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm; boxed region: 12 × 12 μm. (B) Time-course plot showing changes in cytoplasmic actin (mApple-F-tractin) following histamine addition. Y-axis values represent fluorescence intensity at each time point, normalized to the maximum fluorescence intensity observed (20 sec time point in INF2 WT). Error bars represent the standard error of the mean (SEM) of the indicated cell numbers. (C, D) Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing either GFP-INF2-CAAX WT (C) or GFP-INF2-CAAX R218Q (D), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Expressing, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Western blots of total cell lysate and LSP from U2-OS GFP-INF2-CAAX stable cells transfected with either control siRNA or CAP1/2 siRNAs. Equal protein (5 μg) was loaded in each lane and blotted with the indicated antibodies. (B, C) Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS GFP-INF2-CAAX cells transfected with either control siRNA (B) or CAP1/2 siRNAs (C), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, 1 μM calcium with 100 nM CALM). Experiments were performed four times with similar results, and the data shown are a representative experiment. (D) Pyrene-actin polymerization assay performed as in (B) and (C), without siRNA but including 1 μM recombinant CAP2 protein where indicated. Experiments were performed twice with similar results. Data shown are a representative experiment. (E) Time-lapse montage of U2-OS cells transfected with either control siRNA or CAP1/2 siRNAs, further transfected with mApple-F-tractin, and stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm; boxed region: 12 × 12 μm. (F) Time-course plot showing changes in cytoplasmic actin intensity (mApple-F-tractin) following histamine addition in U2-OS control KD cells and CAP1/2 KD cells. Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F₀). Error bars represent the standard error of the mean (SEM), of the stated number of cells analyzed. (G, H) U2-OS cells transfected with either control siRNA or CAP1/2 siRNA were fixed and stained with TRITC-phalloidin without stimulation (G), or with 100 μM histamine treatment for 30 seconds (H). Insets show magnified views of the boxed regions, as indicated by the corresponding numbers. N =nucleus. Scale bar: 20 μm; boxed region: 20 × 20 μm. (I, J) Quantification of actin filament intensity in the perinuclear region without stimulation (H) or with 100 μM histamine treatment for 30 seconds (I), normalized to nuclear intensity. Each point represents one region of interest (ROI) per cell. P -value was calculated from an unpaired t- test. n.s. =not significant. error bars represent the standard error of the mean (SEM). Experiments were performed in triplicate.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Western Blot, Transfection, Control, Labeling, Polymerization Assay, Recombinant, Fluorescence, Staining

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: B) Pyrene-actin polymerization assay containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, and 10 nM INF2 full-length (FL) protein, with the indicated concentrations of thymosin (A). Actin polymerization rates were calculated from the linear phase (0-250 seconds) of the pyrene-actin assay (B). Experiments were performed twice with similar results, and the data shown are a representative experiment. (C, D) Same as (A) and (B), but performed in the presence of 2 μM profilin. (E, F) Same as in (A), but performed in the presence of 10 nM INF2-FFC, 2.9 μM thymosin, 6 μM profilin and the indicated concentrations of INF2-DID (DID). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (G, H) Pyrene-actin polymerization assay containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 2.9 μM thymosin, 6 μM profilin, 1 μM calcium and either 10 nM INF2 full-length (G) or 10 nM INF2-FFC with 300 nM INF2-DID (H), in the presence of the indicated concentrations of CALM. (I) Calculations of actin polymerization rates for CALM concentration curves from experiments with INF2-FL (top) or INF2-FFC + INF2-DID (bottom).

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Polymerization Assay, Labeling, Pyrene Actin Assay, Concentration Assay

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: Extended data for figure 6. (A-C) Pyrene-actin polymerization assays containing 2 µM actin (5% pyrene-labeled), 50 nM capping protein, and 10 nM INF2-FFC were performed with the following conditions: varying concentrations of thymosin alone (A), varying thymosin plus 2 µM profilin (B), or with/without INF2-DID (C). All experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (D) Actin polymerization rates from INF2-FFC inhibition assays in the presence of 2.9 μM thymosin, 6 μM profilin, 50 nM INF2-DID, and varying CALM (1 μM free calcium). (E-G) Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, and 10 μg of LSP from U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX were conducted with: varying concentrations of thymosin alone (A), or varying thymosin plus 0.25 μM profilin (B). Actin polymerization rates were calculated from the linear phase of the pyrene-actin polymerization curve (C). (H, I) Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 2.5 μM thymosin, 0.25 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX were performed in the presence or absence of: varying concentrations of anti-DID antibody (D) or CALM in the presence of 1 μM free calcium (E). All experiments were performed twice with similar results, and the data shown are representative of all results.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Labeling, Inhibition, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: Pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled) and 10 μg of LSP from U2-OS INF2 KO cells stably expressing GFP-INF2-CAAX were performed in the presence of (A) 68 nM capping protein, 2.4 μM thymosin and 0.53 μM profilin, (B) 102 nM capping protein, 3.6 μM thymosin and 0.795 μM profilin, or (C) 136 nM capping protein, 4.8 μM thymosin and 1.06 μM profilin, with the indicated components (330 nM anti-DID antibody, or 1 μM calcium with 100 nM CALM). All experiments were performed in triplicate with similar results, and the data shown are representative of all results. See Materials and Methods for calculation of these protein levels.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Labeling, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Fluorescence anisotropy assay to measure actin monomer or INF2 DID binding by the INF2 C-terminal region or mDia1 DAD peptide. FITC-labeled INF2 C-terminal or TAMRA-labeled mDia1-DAD peptide (50 nM) were incubated with varying concentrations of LatA-stabilized actin or INF2 DID. (B) Pyrene-actin assays containing 2 μM actin monomers (5% pyrene labeled) and 2.5, 5, or 10 nM of the FFC construct of WT INF2 or the INF2-mDia1 DAD chimera. (C) Images from live-cell experiments in which U2-OS INF2 KO cells were transfected with mApple-F-tractin and either GFP-INF2 CAAX WT or GFP-INF2 CAAX-mDia1-DAD chimera. Cells stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm; boxed region: 12 × 12 μm. (D) Time-course plot showing changes in cytoplasmic actin intensity (mApple-F-tractin) following histamine addition in U2-OS INF2 KO cells expressing either GFP-INF2 CAAX-WT or GFP-INF2 CAAX-mDia1-DAD. Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F₀). Error bars represent the standard error of the mean (SEM), of the stated number of cells analyzed. (E, F) U2-OS INF2 KO cells transfected with either GFP-INF2 CAAX-WT or GFP-INF2 CAAX-mDia1 DAD were fixed and stained with TRITC-phalloidin without stimulation (D), or with 100 μM histamine treatment for 30 seconds (E). Insets show magnified views of the boxed regions, as indicated by the corresponding numbers. N =nucleus. Scale bar: 10 μm; boxed region: 15 × 15 μm. (G, H) Quantification of actin filament intensity in the perinuclear region without stimulation (F) or with 100 μM histamine treatment for 30 seconds (G), normalized to nuclear intensity. Each point represents one region of interest (ROI) per cell. P -value was calculated from an unpaired t- test. n.s. =not significant. error bars represent the standard error of the mean (SEM). (I) Western blot of LSP from U2-OS INF2 KO cells transiently expressing either GFP-INF2-CAAX WT or GFP-INF2 CAAX-mDia1-DAD cells. Equal protein (5 μg) was loaded in each lane and blot was probed with the indicated antibodies. (J, K) LSP pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type (J) or GFP-INF2 CAAX-mDia1 DAD (K), with the indicated components (330 nM anti-DID antibody or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Fluorescence, Binding Assay, Labeling, Incubation, Construct, Transfection, Expressing, Staining, Western Blot

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Fluorescence anisotropy assay to measure CALM binding by the INF2 N-term peptide (1-29 aa). FITC-labeled INF2 N-term was incubated with varying concentrations of CALM. Two conditions were used: Condition 1 was 100 nM FITC-INF2-NT and 50 μM free calcium (blue); Condition 2 was 10 nM FITC-INF2-NT and 1 μM free calcium (red). The K d app were 4.5 and 3.6 μM, respectively. (B) Images from a time-lapse movie of U2-OS INF2 KO cells transiently expressing mApple-F-tractin along with either GFP-INF2-CAAX wild-type, WLL or ΔNT mutant. Cells were stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm; boxed region: 12 × 12 μm. (C, D) Time-course plot showing changes in cytoplasmic actin (mApple-F-tractin) following histamine addition in GFP-INF2-CAAX wild-type cells, WLL mutant (C) or ΔNT mutant (D). Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F₀). Error bars represent the standard error of the mean (SEM) for indicated number of cells. (E-G) Cell-free pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type (E), WLL mutant (F) or ΔNT mutant (G), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment. (H, I) Cell-free pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type (H), or ΔNT mutant (I), with the indicated components (5 μM or 10 μM INF2 N-terminal peptide).

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Fluorescence, Binding Assay, Labeling, Incubation, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Western blots of total cell lysate from U2-OS INF2 KO cells transiently expressing the indicated GFP or GFP-INF2-CAAX constructs. Cells were transfected, and total protein was extracted 24 hours post-transfection. (B) Live-cell imaging of U2-OS INF2 KO cells co-transfected with GFP-INF2-CAAX constructs and ER-RFP to assess the subcellular localization of INF2 variants. Scale bar: 10 μm.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Western Blot, Expressing, Construct, Transfection, Live Cell Imaging

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Time-course plot showing changes in cytoplasmic actin (mApple-F-tractin) following histamine addition in GFP-INF2-CAAX wild-type or INF2 N-terminal mutants. Y-axis values represent fluorescence intensity at each time point normalized to time 0 (F/F₀). Experiments were performed in triplicate; error bars represent the standard error of the mean (SEM). (B) Fluorescence anisotropy competition assays with 100 nM FITC-INF2-NT, 2 μM CALM, 50 μM free calcium, and a range of concentrations of the indicated INF2 construct (C) Fluorescence anisotropy assay with 50 nM FITC-INF2 C-terminal region (891-1250 aa) and a range of concentrations of the indicated INF2 DID regions. (D) Alignment of INF2 N-terminal sequences across various species. Mutants used in this study are indicated above the aligned sequences. Asterisks (*) indicate identical residues. Sequence alignment was performed using COBALT, with Rasmol color coding for amino acids. Species (with UniProt numbers): H. sapiens (human, Q27J81), P. abelii (orangutan, H2NME2), M. musculus (mouse, Q0GNC1), E. caballus (horse, F6UX87), B. taurus (cow, A0AAA9SIE1), P. macrocephalus (sperm whale, A0A2Y9TAJ8), M. domestica (opossum, F6TXY8), P. ornithorhynchus (platypus, A0A6I8NFG2), G. gallus (chicken, A0A8V0Z7F3), X. tropicalis (frog, A0A6I8SNL0), L. oculatus (fish, W5N4V5). (E, F) AlphaFold model of full-length INF2-CAAX, generated in AlphaFold3. Residue A149 in DID and L976 and L977 in DAD shown in red. (G, H) AlphaFold model of a human construct lacking the DAD ( https://alphafold.ebi.ac.uk/entry/A0A087X118 ), in which the N-terminal peptide interacts with the DAD-binding region of the DID, albeit in a diserent orientation. Residue A149 in DID shown in red, and W11 in N-terminus shown in blue. In both models, residues outside of the N-term (purple, 1-34), DID (green, 35-263) and DAD (blue, 972-990) have been omitted. Top: full model. Bottom: zoom to region of interest.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Fluorescence, Construct, Sequencing, Generated, Residue, Binding Assay

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: (A) Time-lapse montage of U2-OS INF2 KO cells transiently expressing mApple-F-tractin along with either GFP-INF2-CAAX wild-type, NM or LM mutant. Cells were stimulated with 100 μM histamine at time 0. Insets show magnified views of the boxed regions. Scale bar: 10 μm; boxed region: 12 × 12 μm. (B) Time-course plots showing changes in cytoplasmic actin filaments (mApple-F-tractin) following histamine addition in GFP-INF2-CAAX wild-type, NM or LM mutant expressing cells. Y-axis values represent fluorescence intensity at each time point, normalized to the maximum fluorescence intensity observed. Error bars represent the standard error of the mean (SEM) of the indicated cell numbers. (C) U2-OS INF2 KO cells transiently expressing GFP-INF2-CAAX wild-type, NM or LM mutant were fixed and stained with TRITC-phalloidin without stimulation. Insets show magnified views of the boxed regions. N =nucleus. Scale bar: 20 µm; boxed region: 20 × 20 µm. (D) Quantification of actin filament intensity in the perinuclear region, normalized to nuclear intensity. Each point represents one region of interest (ROI) per cell. P -value calculated from an unpaired t- test. Error bars represent the standard error of the mean (SEM). (E,F) Cell-free pyrene-actin polymerization assays containing 2 μM actin (5% pyrene-labeled), 50 nM capping protein, 6 μM profilin, and 10 μg of LSP from U2-OS INF2 KO cells transiently expressing either GFP-INF2-CAAX wild-type (E), or LM mutant (F), with the indicated components (330 nM anti-DID antibody, 209 nM anti-FH1FH2 antibody, or 1 μM calcium with 100 nM CALM). Experiments were performed in triplicate with similar results, and the data shown are a representative experiment.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Expressing, Mutagenesis, Fluorescence, Staining, Labeling

Journal: bioRxiv

Article Title: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

doi: 10.1101/2025.10.06.680766

Figure Lengend Snippet: INF2 (a dimer) is autoinhibited by the DID/DAD interaction, which also requires actin monomers to be bound by monomer-binding proteins like profilin or thymosin. Increased calcium enables calcium-calmodulin (CALM) binding to INF2’s N-terminus, which disrupts the DID/DAD interaction. Actin monomer binding by the DAD keeps INF2 in the activated state.

Article Snippet: Polyclonal antibody against mouse INF2 (Q0GNC1) N-terminal region (containing the DID) amino acids 1-424 was raised in rabbits (Cocalico Biologicals) and asinity purified by using the antigen coupled to Sulfolink (Pierce, Thermo Fisher Scientific, Rockford, IL).

Techniques: Binding Assay