Axonemes from wild-type and mutant strains were isolated following a published protocol (Craige et al., 2013 ). Briefly, cells were deflagellated with 25 mM dibucaine (Sigma-Aldrich) in HMDS buffer (10 mM HEPES, 5 mM MgSO4, 1 mM DTT, 4% Sucrose, pH 7.4) and centrifugated at 1,800 × g for 5 min to remove cell bodies. The supernatant containing flagella was collected and laid over HMDS buffer containing 25% sucrose. After centrifugation at 2,400 × g for 10 min, the supernatant containing flagella was collected down to the sucrose interface. To remove membranes and matrix from flagella, NP-40 (USB Chemicals) was added to flagella to a final concentration of 1% and centrifuged at 30,000 × g for 20 min. The white pellet at the bottom of tube (representing the axonemes) was resuspended with HMDEKP buffer (30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, PH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences). In the last step, axonemes (equal to 32 OD280) were mildly digested with 10 μg/ml subtilisin A (Sigma-Aldrich) on ice for 30 min in the presence of 2 mM ATP in a total reaction volume of 10 μl before plunge freezing. ProteaseArrest protease inhibitors were present in the buffer to minimize the effects of subtilisin digestion.
Dibucaine
Manufactured by Merck Group
Sourced in United States
Dibucaine is a topical anesthetic agent used in pharmaceutical applications. It functions as a local anesthetic by inhibiting the initiation and transmission of nerve impulses.
Automatically generated - may contain errors
Lab products found in correlation
Proteasearrest protease inhibitors, by G Biosciences (2 mentions)
Subtilisin a, by Merck Group (2 mentions)
Tumor necrosis factor α tnf α, by R&D Systems (2 mentions)
Lipofectamine reagent, by Promega (2 mentions)
Caspase 3 inhibitor, by Merck Group (2 mentions)
Pd98059, by Merck Group (2 mentions)
Penicillin, by Corning (2 mentions)
Acetonitrile, by Vetec (1 mentions)
Triethylamine, by Vetec (1 mentions)
Poloxamer 188 f68, by Merck Group (1 mentions)
96 well microtitre plate, by Corning (1 mentions)
Np 40 alternative, by Merck Group (1 mentions)
P9599, by Merck Group (1 mentions)
Hygromycin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Staurosporin, by Merck Group (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
13 protocols using dibucaine
1
Isolation and Subtilisin Digestion of Axonemes
2
Characterization of Lipid Membrane Probes
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Dibucaine, poloxamer 188 (F68), and 5-doxyl-stearic acid spin labels (5-SASL) were obtained from Sigma-Aldrich (St Louis, MO, USA). Myristyl myristate (MM) and cetyl palmitate (CP) were from Dhaymers Fine Chemicals (São Paulo, Brazil) and Croda (São Paulo, Brazil), respectively. Other reagents used were HPLC grade: acetonitrile (J.T. Baker, Goias, Brazil), triethylamine (Vetec Rio de Janeiro, Brazil), and orthophosphoric acid (Cetus Ind. Com. Prod. Quim., Santo Amaro, Brazil). Deionized water (18.2 mΩ cm) was obtained from a Waters ultrapure water system (Merck KGaA, Darmstadt, Germany).
3
Dibucaine Sensitivity Assay on LB Agar
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Sensitivity to the membrane perturbant dibucaine was assessed on LB agar plates. Briefly, 4-ml cultures were inoculated with overnight cultures at 1:100 dilution and grown in LB at 37°C until an OD600 of about 0.5 was reached. Cell counts were normalised according to OD600, then serially diluted in LB with seven 10-fold dilutions using 96-well microtitre plates (Corning). Two microlitres of the diluted cultures were manually spotted onto the LB agar plates and incubated overnight at 37°C. When indicated, dibucaine (Sigma-Aldrich) was added to the LB agar plate at a final concentration of 1.2 mM.
4
Isolating Cilia for Biochemical Analysis
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To isolate cilia for biochemical analysis, a protocol described by Witman (1986) (link) was used. In brief, cells were concentrated and washed with 10 mM Hepes, pH 7.4. Cells were resuspended in HMS (10 mM Hepes, 5 mM MgSO4, and 4% sucrose) and deciliated by adding dibucaine to a final concentration of 4.17 mM (Sigma-Aldrich). Cilia and cell bodies were separated by differential centrifugations and cilia were collected by sedimentation (17,000 g, 4°C, 20 min). Isolated cilia were resuspended in HMEK (30 mM HEPES, 5 mM MgSO4, 0.5 mM EGTA, and 25 mM KCl) plus protease inhibitor (P9599; 1:100; Sigma-Aldrich) and demembranated by addition of 1% NP-40 Alternative (final concentration; EMD Millipore) on ice for 20 min. Axonemes were pelleted by centrifugation (30,000 g, 4°C, 20 min) and fractions were analyzed by SDS-PAGE and Western blotting.
5
Genetic Modifications in M. smegmatis
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Markerless, knock-in M. smegmatis strains expressing both HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc or HA-mCherry-GlfT2 alone were previously established (Hayashi et al., 2016 (link)). M. smegmatis mc2155 (wild-type), ∆ponA2, and ∆ponA2 L5::ponA2 (wild-type and various alleles of ponA2) were grown in Middlebrook 7H9 growth medium (BD Difco, Franklin Lakes, NJ) supplemented with 11 mM glucose, 14.5 mM NaCl, 0.4% (vol/vol) glycerol, and 0.05% (vol/vol) Tween-80 (Sigma–Aldrich, St. Louis, MO), as well as kanamycin (50 μg/mL) and/or hygromycin (50 μg/mL) where appropriate. Bacteria were grown at 37°C with shaking at 130 rpm. For chemical treatments, 200 µL of 5 M benzyl alcohol in DMSO (Sigma-Aldrich) or 20 µL of 0.2 M dibucaine in water (Sigma-Aldrich) was added to a 10 mL log-phase culture to achieve a final concentration of 100 mM or 0.4 mM, respectively,. The same volume of DMSO (200 μL to 10 mL culture, 2% [v/v]) or water (20 μL for 10 mL culture) was added as negative control. Phosphate-buffered saline (PBS) with 0.05% (vol/vol) Tween-80 (PBST) was used to wash out benzyl alcohol prior to resuspending bacteria in Middlebrook 7H9.
6
Isolation and Dissociation of Chlamydomonas Axonemes
7
Mycobacterium smegmatis Stress Response
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M. smegmatis mc2155 was grown at 37°C in Middlebrook 7H9 broth supplemented with 11 mM glucose, 14.5 mM NaCl, and 0.05% (vol/vol) Tween 80. Dibucaine (Sigma-Aldrich) was added to a log-phase culture at the final concentration of 200 μg/mL, and an equivalent volume of water was used as a vehicle control. After a 3-h incubation at 37°C, the culture was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 80 (PBST) three times and resuspended in the same volume of Middlebrook 7H9 broth for recovery. Benzyl alcohol treatment was identical to Dibucaine treatment except that the treatment was for 1 h at the final concentration of 100 mM, as described previously (14 (link)). CFU were determined by serially diluting cell culture using Middlebrook 7H9 broth and spotting 5 μL on Middlebrook 7H10 agar supplemented with 11 mM glucose and 14.5 mM NaCl. The agar plates were incubated at 37°C for 2 to 3 days before the number of microcolonies was determined.
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8
Reagents for Cell Culture and Cytotoxicity Assays
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Dulbecco's Modification of Eagle's Medium (DMEM) with 4.5 g/L glucose, l -glutamine and sodium pyruvate and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin; 1% P/S). were obtained from Corning (Mediatech, Inc. Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Lipofectamine reagent was from Promega (Madison, WI, USA). Amphotericin B (fungizone), Geneticin (G418), Bay K 8644, wortmannin, PD98059, staurosporin, dibucaine, caspase-3 inhibitor, lipopolysaccharide (LPS) and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tumor necrosis factor-α (TNF-α) was purchased from R&D Systems (Minneapolis, MN, USA). caspase-3 inhibitor was diluted in phosphate buffered saline (PBS) and other reagents were dissolved in 100% ethanol before use.
9
Isolation and Purification of Axonemal Dyneins
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As described in previous work (29 (link), 30 ), the oda2-t-lc2-bccp cells were harvested and the axonemes were isolated by standard methods (34 (link)). Briefly, cells were harvested by centrifugation and deciliated with dibucaine (Sigma-Aldrich, St. Louis, MO). The cilia were isolated and diluted into HMDE (30 mM HEPES, 5 mM MgSO4, 1 mM DTT, and 1 mM EGTA, titrated to pH 7.4 with potassium hydroxide (KOH)) with 0.4 mM Pefabloc (Sigma-Aldrich). They were demembranated with IGEPAL CA-630 (Sigma-Aldrich) and washed in HMDE. The axonemal dyneins were extracted with 0.6 M KCl and purified using a MonoQ 10/100 GL (GE Healthcare, Piscataway, NJ) ion-exchange column (35 ). The axonemal dyneins were eluted with a linear gradient of 150 to 400 mM KCl in HMDE and stored at 300 μg/mL in 30% saturated sucrose at -80° C.
10
Isolation and Analysis of Flagella Proteins
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To isolate flagella for protein analysis, we followed a protocol described by Witman (1986) (link). In brief, cells were concentrated and washed in 10 mM HEPES, pH 7.4, resuspended in HMS at 4°C (10 mM HEPES, 5 mM MgSO4, and 4% sucrose) and deflagellated by adding dibucaine to a final concentration of 4.17 mM (Sigma-Aldrich) and repeated pipetting. Flagella were separated from cell bodies by centrifugation, collected from the supernatant by centrifugation, and resuspended in HMEK (30 mM HEPES, 5 mM MgSO4, 25 mM KCl, and 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA]) with protease inhibitor cocktail (Sigma-Aldrich). For Western blotting, SDS–PAGE sample buffer was added to flagella samples and samples were incubated at 85°C for 10 min. The following primary antibodies were used: mouse anti-IC2 (1:1000; King and Witman, 1990 (link)), mouse anti-IFT81 (1:1000; Cole et al., 1998 (link)), rabbit anti-ODA16 (1:200; Ahmed and Mitchell, 2005 (link)), and mouse anti-NG (1:1500; Chromotek). Western blots were developed using anti-mouse and anti-rabbit immunoglobulins G conjugated to horseradish peroxidase (Invitrogen) and chemiluminescence substrate (Michigan Diagnostics). Images were captured using a BioRad Gel Doc imaging system.
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