Blebbistatin

Manufactured by Cell Signaling Technology

Blebbistatin is a small molecule compound that specifically and reversibly inhibits non-muscle myosin II ATPase activity. It is a useful tool for studying the role of non-muscle myosin II in cellular processes.

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Lab products found in correlation

3 protocols using blebbistatin

Characterization of Neisseria gonorrhoeae antibodies

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The purified chicken IgY anti-Neisseria gonorrhoeae antibody was raised against formalin-killed FA1090 gonococci by Cocalico Biologicals and it cross-reacts with the MS-11, FA19 and F62 strains. The rabbit anti-N.g polyclonal antibody (cat# 20-NR08) was purchased from Fitzgerald Industries. Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling—Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S). Mouse antibodies against the following antigens were purchased from Santa Cruz–Actin (sc8432), FMNL1 (sc390466), FMNL2 (sc390298), FHOD1 (sc365473), DAAM1 (sc100942). Mouse anti-Cas9 (7A9-3A3) was purchased from Cell Signaling. Anti-FMNL3 (ab57963) and anti-Galectin 3 (ab2785) mouse monoclonal antibodies were purchased from Abcam. Anti-human LAMP-2 (H4B4) was purchased from BioLegend. Anti-DIAPH2 (A300-079A-T) was purchased from Bethyl Labs. Highly cross-absorbed Alexa fluorophore conjugated secondary antibodies were purchased from Life Technologies.
Inhibitors used in this study were purchased from: (1) Cayman Chemicals—cytochalasin D (5μM), CK869 (10μM), Dynasore (30μM), GSK269962 (1μM), Nocodazole (3μM), ML-141 (5μM), EHT 1864 (5μM), BMS-5 (1μM) and (-) blebbistatin (5μM); (2) Cell Signaling—LY294002 (10μM); (3) Abcam—SMIFH2 (12.5μM to 100μM).

Ivanov S.S., Castore R., Juarez Rodriguez M.D., Circu M, & Dragoi A.M. (2021). Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages. PLoS Pathogens, 17(12), e1010184.

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Acto-myosin inhibition in T-cell spreading

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T-cell spreading assay was performed at 1×10⁶ cells/ml concentration. Cells treatment with non-enantiomeric (−/−)-Blebbistatin (Sigma) was performed in two different manners: For immunofluorescent analysis the treatment was continuous throughout pretreatment to the cell spreading stages: 15 minutes prior to the cells plating samples were resuspended in 100 μM Blebbistatin solution in supplemented complete RPMI media and incubated on micro-chips. For live cell imaging of acto-myosin inhibition, Blebbistatin was added at the moment of interest to its final concentration of 100 μM. Calyculin A (Cell Signaling Technology, Inc), was added to the working concentration of 50 nM at the moment of interest. For formin and ARP2/3 inhibition experiments, cells were treated with 5 μM smiFH2 and 50 μM CK-666, respectively (both Sigma) for 15 minutes prior to cell plating and spread in presence of the drugs. For FRAP experiments, smiFH2 and ARP2/3 were added 30 minutes after plating and cells were imaged immediately after drug addition. Immunofluorescence staining was performed as described previously⁶⁰.

Tabdanov E., Gondarenko S., Kumari S., Liapis A., Dustin M.L., Sheetz M.P., Kam L.C, & Iskratsch T. (2015). Micropatterning of TCR and LFA-1 ligands reveals complementary effects on cytoskeleton mechanics in T-cells. Integrative biology : quantitative biosciences from nano to macro, 7(10), 1272-1284.

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Antibodies and Inhibitors for Gonococcal Trafficking

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The purified chicken IgY anti-Neisseria gonorrhoeae antibody was raised against formalin-killed FA1090 gonococci by Cocalico Biologicals and it cross-reacts with the MS-11, FA19 and F62 strains. The rabbit anti-N.g polyclonal antibody (cat# 20-NR08) was purchased from Fitzgerald Industries. Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling -Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S).
Mouse antibodies against the following antigens were purchased from Santa Cruz -Actin (sc8432), FMNL1 (sc390466), FMNL2 (sc390298), FHOD1 (sc365473), DAAM1 (sc100942).
Mouse anti-Cas9 (7A9-3A3) was purchased from Cell Signaling. Anti-FMNL3 (ab57963) and anti-Galectin 3 (ab2785) mouse monoclonal antibodies were purchased from Abcam. Anti-human LAMP-2 (H4B4) was purchased from BioLegend. Anti-DIAPH2 (A300-079A-T) was purchased from Bethyl Labs. Highly cross-absorbed Alexa fluorophore conjugated secondary antibodies were purchased from Life Technologies.
Inhibitors used in this study were purchased from: (1) Cayman Chemicals -cytochalasin D (5µM), CK869 (10µM), Dynasore (30µM), GSK269962 (1µM), Nocodazole (3µM), ML-141 (5µM), EHT 1864 (5µM), BMS-5 (1µM) and (-) blebbistatin (5µM); (2) Cell Signaling -LY294002 (10µM); (3) Abcam -SMIFH2 (25µM).

Ivanov S.S., Castore R., Circu M.L., & Dragoi A. (2021). Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages.

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