G-Actin (Life Technologies) was polymerized for 2.5 h at 25°C in polymerization buffer (5 mM Tris pH 8.0, 0.2 mM CaCl2, 1mM ATP, 0.5 mM DTT, 5 mM KCl, 2 mM MgCl2) containing equal amounts (0.125 μg/μl) of either only G-Actin or G-Actin and recombinant Simiate, or a control protein (GST), respectively. Following ultracentrifugation at 100,000 g for 1 h to sedimentate F-Actin, the supernatant as well as the pellet were subjected to SDS-PAGE and western blotting to analyze the distribution of each protein by Ponceau staining or directly following SDS-PAGE by Coomassie staining, respectively. Alternatively, Actin was pre-polymerized, sedimentated, and subsequently incubated with Simiate or the control protein as outlined above. For further information on the interpretation, please also see the chapter “Bioinformatics and Statistics.”
G actin
Manufactured by Thermo Fisher Scientific
Sourced in United States
G-actin is a globular protein that is the monomeric subunit of the cytoskeletal protein actin. It plays a fundamental role in various cellular processes such as cell motility, cell division, and the maintenance of cell shape.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
F actin, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mrtf a, by Santa Cruz Biotechnology (1 mentions)
Anti mrtf a, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated dnase 1, by Thermo Fisher Scientific (1 mentions)
Ix51 microscope, by Zeiss (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl system, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Fluorsavetm, by Merck Group (1 mentions)
Axio observer z1, by Nikon (1 mentions)
7 protocols using g actin
1
Actin Polymerization Assay with Simiate
2
GUV Formation with Encapsulated Actin
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DOPC and ATTO 655 DOPE were mixed
in a 99.9:0.1 molar ratio to prepare the lipid-in-oil dispersion.
100 μL of G-actin (4.4 μM, 9% labeled with Alexa Fluor
488) in G-buffer (5 mM Tris-HCl pH 7.8, 0.1 mM CaCl2, 0.02
mM ATP and 4 mM DTT) and 18.5% v/v OptiPrep was encapsulated in every
experiment, only varying rotation speed and capillary size. For a
capillary size of 25 μm, the flow rate was lowered to 2.5 μL
min–1 to reduce the pressure in the capillary setup.
The encapsulated volume was reduced to 50 μL in these experiments.
GUVs were produced in an outer aqueous solution containing approximately
85 mM glucose in Milli-Q water. G-actin was purchased from Hypermol
and Alexa Fluor 488-labeled G-actin was obtained from Invitrogen.
All proteins were handled according to instructions provided by the
manufacturer. GUVs were imaged in the outer aqueous solution using
confocal microscopy, 50 μL of GUV solution was deposited on
a custom-made glass coverslip and covered. Microscopy was performed
using a Nikon A1R-MP confocal microscope, using a Plan APO IR 60×
water immersion objective. The 561 nm (laser power 1.0) and 488 nm
(laser power 1.0) laser lines were used in combination with the appropriate
emission filters to image the ATTO 655-labeled DOPE membrane and Alexa
Fluor 488-labeled G-actin, respectively.
in a 99.9:0.1 molar ratio to prepare the lipid-in-oil dispersion.
100 μL of G-actin (4.4 μM, 9% labeled with Alexa Fluor
488) in G-buffer (5 mM Tris-HCl pH 7.8, 0.1 mM CaCl2, 0.02
mM ATP and 4 mM DTT) and 18.5% v/v OptiPrep was encapsulated in every
experiment, only varying rotation speed and capillary size. For a
capillary size of 25 μm, the flow rate was lowered to 2.5 μL
min–1 to reduce the pressure in the capillary setup.
The encapsulated volume was reduced to 50 μL in these experiments.
GUVs were produced in an outer aqueous solution containing approximately
85 mM glucose in Milli-Q water. G-actin was purchased from Hypermol
and Alexa Fluor 488-labeled G-actin was obtained from Invitrogen.
All proteins were handled according to instructions provided by the
manufacturer. GUVs were imaged in the outer aqueous solution using
confocal microscopy, 50 μL of GUV solution was deposited on
a custom-made glass coverslip and covered. Microscopy was performed
using a Nikon A1R-MP confocal microscope, using a Plan APO IR 60×
water immersion objective. The 561 nm (laser power 1.0) and 488 nm
(laser power 1.0) laser lines were used in combination with the appropriate
emission filters to image the ATTO 655-labeled DOPE membrane and Alexa
Fluor 488-labeled G-actin, respectively.
3
Actin Cytoskeleton Visualization in hCASMCs
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hCASMCs were seeded onto glass coverslips in M199 medium. Sub-confluent cells were starved and pretreated with NGR1, and then stimulated with 5% FBS for 16 h. Cells were fixed with 4% PFA for 10 min, permeabilized in 0.1% Triton X-100 in PBS for 5 min at RT, and stained with Alexa-488-tagged phalloidin (F-actin, Invitrogen) and Alexa-594-tagged DNaseI (G-actin, Invitrogen) for 30 min at RT. The nucleus was stained with DAPI. Images of cells were acquired using a ZEISS LSM 710 confocal microscope. Confocal images of the basal plane of phalloidin-labeled hCASMC were taken using the same gain and offset settings. Each group was quantified twenty cells and the average pixel intensity of each cell was quantified by image J.
4
Immunofluorescence Analysis of Mechanosensitive Proteins
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Cells were fixed in 4% paraformaldehyde in PBS solution for half an hour. Fixed cells were then permeabilized with 0.03% Triton-X and blocked in BSA and goat serum or fish block (for MRTF-A staining) for 1 h. Primary antibodies were incubated overnight at 4 °C with gentle agitation (anti-MRTF-A, Santa Cruz, Dallas, TX, USA, sc-21558; anti-YAP, Cell Signaling, Danvers, MA, USA 14074; anti-PPARγ, Pierce, Waltham, MA, USA, PA3-821A; anti-αSMA, Sigma, St. Louis, MO, USA, a2547). Primary antibodies were washed with PBS several times prior to incubation with fluorescent secondary antibodies. Counterstains were done with DAPI, to stain nuclei, and TRITC conjugated phalloidin, to stain F-actin (Millipore, Burlington, MA, USA, FAK100) or Alexa Fluor 488-conjugated DNAse-I (Thermo Scientific, Waltham, MA, USA), to preferentially stain G-actin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescent micrographs were taken with an Olympus IX51 microscope (to visualize YAP, MRTF-A, and PPAR-γ) or Zeiss 710 confocal microscope (to visualize F-actin, G-actin, αSMA). Fluorescent intensity was quantified on at least 50 cells per condition, n = 3–4 animal replicates, using ImageJ. DAPI counterstains were used to create masks to identify nuclear regions for YAP and MRTF-A localization for quantitation.
5
Fluorescent Labeling of Cytoskeletal Proteins
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The primary antibodies used in this study are listed in Table S1 . Detection was achieved with goat anti-rabbit, anti-mouse, or anti-rat IgG highly cross-absorbed secondary antibodies coupled to Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 647 (1:600; all from Life Technologies). F-Actin was labeled with either Alexa Fluor 488– or Alexa Fluor 546–phalloidin (1:200; Life Technologies). G-Actin was labeled with Alexa Fluor 488–deoxyribonuclease I (1:500; Thermo Fisher Scientific).
6
Nonglucosylated Rac1 Detection Protocol
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MEM and Dulbecco's modified Eagle medium (DMEM) cell culture medium and fetal calf serum (FCS) were from Invitrogen (Karlsruhe, Germany) and materials for cell culture from TPP (Trasadingen, Switzerland). Complete® protease inhibitor was supplied by Roche (Mannheim, Germany), protein weight marker PageRuler Prestained Protein Ladder® by Thermo Fisher Scientific Inc. (Waltham, MA, USA). Baf A1 and Bac were obtained from Calbiochem (Bad Soden, Germany), and the enhanced chemiluminescence (ECL) system was from Millipore (Schwalbach, Germany). The antibody detecting nonglucosylated Rac1 [26 (link)] was from BD Biosciences (Franklin Lakes, NJ, USA), against G-actin from Thermo Fisher Scientific Inc. (Waltham, MA, USA), and the secondary peroxidase-coupled anti-mouse antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). TcdB was purified as described [13 (link)].
7
Quantifying F-actin and G-actin in hCOs
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F-actin and G-actin measurement was performed as described.65 D28 hCOs were dissociated into single cells using the same method used for scRNA-seq. Harvested cells were resuspended in organoid maturation medium and passed through a 40 μm filter strainer to obtain single cells. Cells then seeded at a density of 0.05 million/well on 24-well size coverslips coated with hESC qualified Matrigel. After two days in culture, cells were fixed with 4% PFA for 15 min. Permeabilization and blocking of the samples are performed as mentioned in the section on immunofluorescence microscopy. After blocking, cells were stained with antibody Alexa Fluor™ 647 Phalloidin (F-actin, ThermoFisher Scientific, Cat#A22287), Deoxyribonuclease I, Alexa Fluor™ 488 Conjugate (G-actin, ThermoFisher Scientific, Cat# D12371) and DAPI (ThermoFisher Scientific, Cat#D1306) for 2 h at room temperature. Cells were then washed thrice in 1×PBS and mounted with FluorSaveTM (Merck, Cat#345789) for imaging (Nikon, Eclipse Ti2; Zeiss, Axio Observer Z1 and Nikon, Eclipse TE2000-E). The same exposure settings were used to acquire images from WT and FEZ1-null groups. Images were analyzed with ImageJ/Fiji with quantification details in the following section. At least three independent experiments were performed.
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