Experiments with ATII cells were performed in bath solution (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 5 glucose, 10 Hepes; pH 7.4). Cells were stimulated for secretion with 100 μM ATP (Sigma, Schnelldorf, Germany). Fusions were detected by staining secretory vesicles with Lysotracker Blue (100 nM) or LysoTracker Red (10 nM) for 20 min (Molecular Probes, Life technologies, Darmstadt, Germany). LysoTracker dyes accumulate in LBs and rapidly diffuse out of the vesicle after fusion52 (link). Microtubule polymerization was inhibited by colchicine (50 µM for 3 h) or nocodazole (60 µM for 30 min), and actin polymerization was inhibited by latrunculin B (10 µM in the experimental bath solution), all from Sigma Aldrich (Steinheim, Germany). Small GTPases were inhibited by B-toxin (300 ng/ml, for 24 h; Merck Millipore, Billerica, USA) and Rho-Inhibitor (2 μg/ml, 12 to 24 h, Cytoskeleton, Inc., Denver, USA).
Rho inhibitor
Manufactured by Cytoskeleton
Sourced in United States
The Rho inhibitor is a laboratory reagent that inhibits the activity of Rho, a family of small GTPase proteins involved in various cellular processes. This inhibitor can be used to study the role of Rho proteins in cellular functions, such as cytoskeletal organization, cell adhesion, and signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Colchicine, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Lysotracker blue, by Thermo Fisher Scientific (1 mentions)
Latrunculin b, by Merck Group (1 mentions)
Pf573228, by Merck Group (1 mentions)
Ml141, by Merck Group (1 mentions)
Tapi 1, by Merck Group (1 mentions)
Gdc 0941, by Genentech (1 mentions)
Ephrinb1 fc, by R&D Systems (1 mentions)
Nsc23766, by Santa Cruz Biotechnology (1 mentions)
Ipa 3, by Merck Group (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Phospho fak, by Thermo Fisher Scientific (1 mentions)
Integrin α5, by Abcam (1 mentions)
Fibronectin, by SCBT (1 mentions)
Integrin α4, by Abcam (1 mentions)
4 protocols using rho inhibitor
1
ATII Cell Exocytosis Regulation
2
Inhibitors for Cell Segregation Screen
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Inhibitors used in cell segregation screen were 2 µg/ml unclustered ephrin-B1-Fc (R&D Systems), 20 µM UO126 (LC Labs), 1 µM PF573228 (Sigma-Aldrich), 1 µM GDC0941 (Genentech), 5 µM IPA3 (Sigma-Aldrich), 10 µM TAPI-1 (EMD Millipore), 10 µM NSC23766 (Santa Cruz Biotechnology, Inc.), 10 µM ML141 (EMD Millipore), 2 µg/ml Rho inhibitor (cell-permeable C3 transferase; Cytoskeleton, Inc.), 20 µM Y-27632 (Cayman), and 25 µM ML7 (EMD Millipore).
3
Rho Inhibitor Effects on Bone Marrow Cells
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Bone marrow cells derived from wild type mice were cultured in Iscove’s medium supplemented with IL7 for 5 days. Cells were serum starved for 3 hours before treating with different concentrations of RHO Inhibitor (CT04, Cytoskeleton, Inc). After 16 hours cells were collected, washed and lysed with RIPA buffer containing Protease and Phosphatase inhibitor cocktail. The amounts of protein in the lysates were determined using Bradford assay and proteins of interest were detected using immunoblotting.
4
Hypoxia Signaling Pathway Regulation
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Antibodies for immunoblotting and immunocytochemistry: GAPDH (Invitrogen, #437000), HIF-1α (Cell Signaling Technology, #14179S), HIF-2α (Abcam, #ab207607), Fibronectin (IB) (Abcam, #ab23750), Fibronectin (ICC) (SCBT, #sc-59826), β-Actin (Invitrogen, #MA1-140), Phospho-FAK (Life Technologies, #396500), Phospho-MYL9 (Invitrogen, #PA5-17727), Total-FAK (Invitrogen, #39-6500), Vinculin (Abcam, #129002), Integrin α4 (Abcam, #ab202969), Integrin α5 (Abcam, #ab150361), GM130 (BD, #610823), Alexa Fluor 568 (Invitrogen #A10037).
Cobalt chloride hexahydrate (MP Biomedicals LLC, #194642) stock solutions were made fresh on the day of the experiment and used at working concentrations of 1 µM, 10 µM and 50 µM. Rho inhibitor (Cytoskeleton, #CT04) was used at a working concentration of 1 µg/ml. Integrin α5 inhibitor ATN-161 (MedKoo Biosciences, #200350a) and Integrin α4 inhibitor BIO5192 (Tocris, #5051) were reconstituted and stored according to manufacturer’s recommendations. DMOG (Abcam, #ab141586) was used at a working concentration of 0.15 mM, 0.25 mM and 0.35 mM for 2 h, 4 h and 6 h.
Cobalt chloride hexahydrate (MP Biomedicals LLC, #194642) stock solutions were made fresh on the day of the experiment and used at working concentrations of 1 µM, 10 µM and 50 µM. Rho inhibitor (Cytoskeleton, #CT04) was used at a working concentration of 1 µg/ml. Integrin α5 inhibitor ATN-161 (MedKoo Biosciences, #200350a) and Integrin α4 inhibitor BIO5192 (Tocris, #5051) were reconstituted and stored according to manufacturer’s recommendations. DMOG (Abcam, #ab141586) was used at a working concentration of 0.15 mM, 0.25 mM and 0.35 mM for 2 h, 4 h and 6 h.
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