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( A ) A549 cells depleted (48 h) by siRNA for Arf, Drp1, or <t>Dnm1</t> were infected (MOI 10, 24 h) with GFP-producing L. pneumophila JR32 or Δ icmT (pNT28), and intracellular replication was assessed by GFP fluorescence using a microtiter plate reader. Graphs show the relative intracellular replication (normalized to GFP signal 1 h p.i.) and represent means + SEM of three independent experiments (Student’s t -test, *, p < 0.05; **, p < 0.01). ( B ) Mitochondria were isolated by differential centrifugation and sucrose density gradient ultracentrifugation from HeLa cells infected (MOI 100, 6 h) with GFP-producing L. pneumophila JR32, ΔicmT , ΔridL , or ΔridL _ ridL (pNT28 or pIF009). Western blot of RidL in cytoplasmic (cyt) and purified mitochondrial (mito) fractions; apoptosis-inducing factor (AIF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a mitochondrial or cytoplasmic marker, respectively. The figure shown is representative for 3 independent experiments. ( C ) HEK293 cells were transfected (24 h) for ectopic production of GFP-RidL (RidL, pKB248), GFP-RidL 9-258 (RidL N , pKB249) or GFP-RidL 259-1167 (RidL C , pKB250), mitochondria were isolated, and the GFP fusion proteins were detected by anti-GFP Western blot in the cell lysate (lys) and purified mitochondrial (mito) fractions. The figure shown is representative for 3 independent experiments. ( D ) Quantification of (C). Signal intensities of GFP and AIF were calculated by Image J. Bars show relative GFP signal in pure mitochondria fractions. Means + SEM of 3 independent experiments are shown (Student’s t- test, **, p < 0.01). ( E, F ) Fluorescence micrographs of HeLa cells infected (MOI 25, 2 h) with mCerulean-producing L. pneumophila JR23, Δ icmT , Δ ridL or Δ ridL_ridL (pNP99 or pKB208). Mitochondria were visualized with MitoTracker Deep Red, and immuno-labelled with specific antibodies against ( E ) Drp1 or ( F ) Tom20 (green) to quantify the localization of these proteins to mitochondria. ( G ) Quantification of signal overlap in (E) and ( H ) quantification of signal overlap in (F) show 75 infected cells from three independent experiments (each dot is a cell; Student’s t -test, ****, p < 0.0001). The brightness of fluorescence signals was linearly increased in Image J for enhanced visibility (A, B); original signal intensities of the images were processed for quantification (C, D).
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( A ) A549 cells depleted (48 h) by siRNA for Arf, Drp1, or Dnm1 were infected (MOI 10, 24 h) with GFP-producing L. pneumophila JR32 or Δ icmT (pNT28), and intracellular replication was assessed by GFP fluorescence using a microtiter plate reader. Graphs show the relative intracellular replication (normalized to GFP signal 1 h p.i.) and represent means + SEM of three independent experiments (Student’s t -test, *, p < 0.05; **, p < 0.01). ( B ) Mitochondria were isolated by differential centrifugation and sucrose density gradient ultracentrifugation from HeLa cells infected (MOI 100, 6 h) with GFP-producing L. pneumophila JR32, ΔicmT , ΔridL , or ΔridL _ ridL (pNT28 or pIF009). Western blot of RidL in cytoplasmic (cyt) and purified mitochondrial (mito) fractions; apoptosis-inducing factor (AIF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a mitochondrial or cytoplasmic marker, respectively. The figure shown is representative for 3 independent experiments. ( C ) HEK293 cells were transfected (24 h) for ectopic production of GFP-RidL (RidL, pKB248), GFP-RidL 9-258 (RidL N , pKB249) or GFP-RidL 259-1167 (RidL C , pKB250), mitochondria were isolated, and the GFP fusion proteins were detected by anti-GFP Western blot in the cell lysate (lys) and purified mitochondrial (mito) fractions. The figure shown is representative for 3 independent experiments. ( D ) Quantification of (C). Signal intensities of GFP and AIF were calculated by Image J. Bars show relative GFP signal in pure mitochondria fractions. Means + SEM of 3 independent experiments are shown (Student’s t- test, **, p < 0.01). ( E, F ) Fluorescence micrographs of HeLa cells infected (MOI 25, 2 h) with mCerulean-producing L. pneumophila JR23, Δ icmT , Δ ridL or Δ ridL_ridL (pNP99 or pKB208). Mitochondria were visualized with MitoTracker Deep Red, and immuno-labelled with specific antibodies against ( E ) Drp1 or ( F ) Tom20 (green) to quantify the localization of these proteins to mitochondria. ( G ) Quantification of signal overlap in (E) and ( H ) quantification of signal overlap in (F) show 75 infected cells from three independent experiments (each dot is a cell; Student’s t -test, ****, p < 0.0001). The brightness of fluorescence signals was linearly increased in Image J for enhanced visibility (A, B); original signal intensities of the images were processed for quantification (C, D).

Journal: bioRxiv

Article Title: The Legionella effector RidL promotes mitochondrial fragmentation through phosphorylation activation of the large GTPase Drp1

doi: 10.1101/2025.10.07.680855

Figure Lengend Snippet: ( A ) A549 cells depleted (48 h) by siRNA for Arf, Drp1, or Dnm1 were infected (MOI 10, 24 h) with GFP-producing L. pneumophila JR32 or Δ icmT (pNT28), and intracellular replication was assessed by GFP fluorescence using a microtiter plate reader. Graphs show the relative intracellular replication (normalized to GFP signal 1 h p.i.) and represent means + SEM of three independent experiments (Student’s t -test, *, p < 0.05; **, p < 0.01). ( B ) Mitochondria were isolated by differential centrifugation and sucrose density gradient ultracentrifugation from HeLa cells infected (MOI 100, 6 h) with GFP-producing L. pneumophila JR32, ΔicmT , ΔridL , or ΔridL _ ridL (pNT28 or pIF009). Western blot of RidL in cytoplasmic (cyt) and purified mitochondrial (mito) fractions; apoptosis-inducing factor (AIF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a mitochondrial or cytoplasmic marker, respectively. The figure shown is representative for 3 independent experiments. ( C ) HEK293 cells were transfected (24 h) for ectopic production of GFP-RidL (RidL, pKB248), GFP-RidL 9-258 (RidL N , pKB249) or GFP-RidL 259-1167 (RidL C , pKB250), mitochondria were isolated, and the GFP fusion proteins were detected by anti-GFP Western blot in the cell lysate (lys) and purified mitochondrial (mito) fractions. The figure shown is representative for 3 independent experiments. ( D ) Quantification of (C). Signal intensities of GFP and AIF were calculated by Image J. Bars show relative GFP signal in pure mitochondria fractions. Means + SEM of 3 independent experiments are shown (Student’s t- test, **, p < 0.01). ( E, F ) Fluorescence micrographs of HeLa cells infected (MOI 25, 2 h) with mCerulean-producing L. pneumophila JR23, Δ icmT , Δ ridL or Δ ridL_ridL (pNP99 or pKB208). Mitochondria were visualized with MitoTracker Deep Red, and immuno-labelled with specific antibodies against ( E ) Drp1 or ( F ) Tom20 (green) to quantify the localization of these proteins to mitochondria. ( G ) Quantification of signal overlap in (E) and ( H ) quantification of signal overlap in (F) show 75 infected cells from three independent experiments (each dot is a cell; Student’s t -test, ****, p < 0.0001). The brightness of fluorescence signals was linearly increased in Image J for enhanced visibility (A, B); original signal intensities of the images were processed for quantification (C, D).

Article Snippet: The insert ( Dnm1 ) was amplified from pEGFP-N1-dynamin (Addgene #120313 ) using primers oKA288/oKA289 and Q5 Hot Start High-Fidelity DNA Polymerase (NEB) including Q5 High GC Enhancer.

Techniques: Infection, Fluorescence, Isolation, Centrifugation, Western Blot, Purification, Marker, Transfection