The antibody against pMLC20 was purchased from AbCam (ab2480). CFTR (Inh)-172 was purchased from Sigma and stored as frozen aliquots of 20 mM in DMSO prior to being used at a final concentration of 20 µM. Blebbistatin enantiomers were purchased from Santa Cruz Biotechnology, Inc., reconstituted in DMSO to a stock of 40 mM, and used at a final concentration of 40 µM. Latrunculin A was purchased from Sigma; stocks were made up in DMSO to 2 mM and used at a final concentration of 5 µM. All inhibitor assays included DMSO in media used at a final concentration of 0.1%. Mannitol and NaCl were also purchased from Sigma. TNF-α was purchased from Shenandoah Biotechnology Inc., stored as a stock of 100 µg/ml in double-distilled water (ddH2O) with 0.1% bovine serum albumin (BSA), and used at a final concentration of 30 ng/ml. Actinomycin D was purchased from Sigma; stocks were made at 5 mg/ml in DMSO and used at a final concentration of 5 µg/ml. All drugs were divided into aliquots and stored in −20°C or −80°C according to the manufacturer’s instructions.
Ab2480
Manufactured by Abcam
Sourced in United Kingdom
Ab2480 is a laboratory equipment product. It serves as a core function for scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Hpa019818, by Merck Group (2 mentions)
T6199, by Merck Group (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mannitol, by Merck Group (1 mentions)
Latrunculin a, by Merck Group (1 mentions)
Cftrinh 172, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Af 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Af488, by Abcam (1 mentions)
Ab76020, by Abcam (1 mentions)
Mouse anti flag mab, by Merck Group (1 mentions)
Rabbit anti gapdh mab, by Cell Signaling Technology (1 mentions)
Mouse anti vinculin mab, by Merck Group (1 mentions)
Alexa fluor conjugated secondary ab, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary ab, by GE Healthcare (1 mentions)
Ab92721, by Abcam (1 mentions)
15 protocols using ab2480
1
Inhibitor Assays for Cell Signaling
2
Visualizing Subcellular Protein Localization
3
Antibody Characterization for Cell Biology
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A rabbit anti–l-afadin pAb was prepared as described previously (Mandai et al., 1997 (link)). The Abs listed below were purchased from commercial sources: rabbit anti–α-catenin pAb (C2081; Sigma-Aldrich); mouse anti–α-actinin mAb (A5044; Sigma-Aldrich); rabbit anti–β-catenin pAb (C2206; Sigma-Aldrich); mouse anti-FLAG mAb (F3165; Sigma-Aldrich); rabbit anti-GAPDH mAb (14C10; Cell Signaling Technology); rabbit anti-myosin light chain (phospho S20) pAb (ab2480; Abcam); rabbit anti-sodium potassium ATPase pAb (ab76020; Abcam); rabbit anti-nonmuscle myosin heavy chain II-B pAb (PRB-445P; BioLegend); mouse anti-vinculin mAb (V9264; Sigma-Aldrich); and rat anti–ZO-1 mAb (sc-33725; Santa Cruz Biotechnology). A rat anti–E-cadherin mAb (ECCD2) was a kind gift from M. Takeichi. The HRP-conjugated secondary Abs used for Western blotting were purchased from GE Healthcare. The Alexa Fluor–conjugated secondary Abs used for immunocytochemistry were purchased from Thermo Fisher Scientific.
4
Western Blot Analysis of Signaling Proteins
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The tissue and cell samples were lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Jiangsu KeyGEN BioTECH, Nanjing, China) and the final concentration of lysates was adjusted to 1.5 μg/μl. When running western blots, the sample volume of each well was 10 μl, that is, 15 μg of total protein (15‐well electrophoresis comb, 1.5 mm). Syk (ab40781, Abcam) and p‐Syk (ab58575, Abcam) antibodies for human tissues and Syk (ab40781, Abcam), p‐Syk (ab58575, Abcam), MLCK (ab76092, Abcam), MLC (ab92721, Abcam), p‐MLC (ab2480, Abcam), ZO‐1 (ab96587, Abcam), and Occludin (ab216327, Abcam) antibodies for mouse tissues were used. The protein bands were visualized by a ChemiDoc Touch Imaging System (Bio‐Rad, USA) and quantified by the Image Lab software. Results were normalized to the GAPDH internal control.
5
Immunofluorescence Microscopy of Ectodermal and Neural Explants
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Ectodermal explants (animal caps) or neural plate explants were dissected from stage 10.5 or 15 embryos and immunofluorescence microscopy was carried out using standard protocols. Briefly, the explants were fixed within MEMFA (4% formaldehyde in 1× MEM salt) at 4 °C for overnight or 2% Trichloroacetic acid (TCA) for 30 min at room temperature and then dehydrated with 100% methanol. The following primary antibodies were incubated after blocking with the filtered 10% goat serum in 1x PBS: Rabbit anti-ephrinBs (1:1,000, 600-401-MP0, Rockland), Mouse anti-Ror2 (1:50, Ror2, DSHB), Mouse anti-C-cadherin (1:50, 6B6, DSHB), Mouse anti-ZO1 (1:250, ZO1-1A12, ThermoFisher), Rabbit anti-pMLC (1:250, ab2480, Abcam), Mouse anti-HA-Alexa Fluor-488/555/647 (1:500, 2-2.2.14, ThermoFisher), Mouse anti-V5-Alexa Fluor-488/555/647 (1:500, 2F11F7, ThermoFisher), and Rabbit anti-GFP-Alexa Fluor-488 (1:500, A-21311, ThermoFisher). The secondary antibodies used were Alexa Fluor-488 or Alexa Fluor-594 conjugated Goat anti-rabbit IgG or anti-mouse IgG (1:500, Invitrogen). For F-actin staining, Alexa Fluor™ 488 Phalloidin (A12379, ThermoFisher). The samples were mounted and imaged using Zeiss LSM880 with Airyscan laser scanning confocal microscope.
6
Immunofluorescent Labeling of Zebrafish Ectoderm
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Zebrafish ectoderm cell aggregates were fixed in 4% paraformaldehyde in PBS for 30 min and per- meabilized in 0.1% Triton X-100 in PBS for 10 min. The non-specific binding sites were blocked by incubation in a culture medium containing 10% serum for 2 h. Primary antibody against phosphorylated (pS19) myosin light chain (rabbit, polyclonal, Anti-MYL12A phospho S19, Abcam ab2480) was applied in 1/100 dilution for 2 h at room temperature and then overnight at 4 °C. Anti-rabbit secondary antibody conjugated with AlexaFluor-555 (Southern Biotech, 4030-32) was used in 1/200 dilution for 4 h at room temperature. All incubations were followed by triple washing steps in PBS for 1 h. Finally, immunolabeled aggregates were mounted on microscopic slides (Thermo Scientific) using a mounting medium (Prolong Glass Antifade Mountant with NucBlue Stain, Invitrogen, P36981) containing NucBlue counterstain to visualize cell nuclei. Fluorescent labels were imaged using a Zeiss Axio Observer Z1 microscope with Zeiss EC Plan-Neofluar 40x/0.75 or Olympus A 100x/1.3 objectives and Zeiss AxioCam MRm CCD camera. Images were processed using NIH ImageJ software.
7
Immunofluorescence Staining of Cytoskeletal Proteins
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Cells were washed, fixed in 4% PFA (Electron Microscopy Science) for 10 min at 4°C, rinsed with PBS, permeabilized with a solution of 0.5% Triton X-100 in PBS for 5 min, incubated for 5 min with 5mM NH4Cl, and blocked in PBS containing 1% BSA (Jackson Immunology) and 1% goat/donkey serum (Sigma-Aldrich) for 30 min at RT. Cells were stained with phalloidin-Alexa Fluor 550 (Molecular Probes), mouse anti–β-catenin (CM1181; ECM Biosciences), rabbit anti–myosin light chain phospho S20 pMLC (ab2480; Abcam) primary antibodies (1 h in blocking buffer at RT), followed by incubation with the appropriate Dylight 480 or 650 secondary antibody (Thermo Fisher Scientific) for 45 min at RT. Coverslips were mounted in Fluoromount medium (Sigma-Aldrich), and fluorescent images were acquired on confocal microscopes (LSM 780 or 710; Zeiss) with a 63×/1.4NA oil-immersion objective.
8
Protein Extraction and Immunoblotting Protocol
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Proteins were extracted with TNTE lysis buffer and immunoprecipitation was performed following standard procedures. Total protein lysates and samples from immunoprecipitation were separated by SDS–PAGE, transferred to nitrocellulose membrane (162-0115, BioRad) and probed with primary antibodies (as listed below) followed by horseradish peroxidase-linked secondary antibodies (NA931 and NA934, GE, 1:10,000). The signals were detected using SuperSignal chemiluminescence reagent (34095, Thermo Scientific). The primary antibodies used for immunoblotting were the following: rabbit anti-Arhgap23 (HPA019818, Sigma-Aldrich, 1:2,000); mouse anti-Flag (F3165 Sigma-Aldrich, 1:5,000); rabbit anti-GAPDH (G9545, Sigma-Aldrich, 1:10,000); mouse anti-αTubulin (T6199, Sigma-Aldrich, 1:10,000); mouse anti-T7 (69522, Novagen, 1:10,000); rabbit anti-RhoA (sc-179, Santa Cruz, 1:500); mouse anti-Rac1 (R56220, Transduction Laboratories, 1:2,000); rabbit anti-phospho-MLC2 (ab2480, Abcam, 1:1,000); and mouse anti-MLC2 (M4401, Sigma-Aldrich, 1:1,000). Full blots are available in Supplementary Fig. 7 .
9
Fluorescent Staining of Fish Keratocytes
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Intracellular components were made visible using fluorescent staining techniques. Briefly, fish keratocytes were rinsed 3 times with PBS (pH ~7.4), fixed and permeabilized with a first incubation of 1 min in 0.035% glutaraldehyde and 0.1% triton X-100 followed by a rinse in PBS and a second 10 min incubation in a 0.14% solution of glutaraldehyde. Coverslips were rinsed extensively with PBS and then incubated for 45 min at 37 °C with Alexa Fluor 488 phalloidin (Invitrogen, 1:200) for staining filamentous actin, DAPI57 (Invitrogen, 1:200) to visualize the nuclei, and a primary antibody (anti-vinculin antibody produced in mouse, Sigma-Aldrich, HVIN-1 clone, 1:200) or anti-myosin light chain antibody produced in rabbit (Abcam, ab2480, 1:300). A tetramethylrhodamine-labelled secondary antibody (goat anti-rabbit 1:400 or goat anti-mouse 1:200, Sigma-Aldrich) was then used for 45 min at 37 °C. Slides were mounted in Slow Fade Gold Antifade (Molecular Probes, Invitrogen).
10
Pericyte Visualization and Kidney Injury Assessment
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Pericytes were labelled by expression of DsRed under control of the NG2 promoter (in mice), or with antibodies to NG2 (1:200; Abcam ab50009, Cambridge, United Kingdom), α-smooth muscle actin (α-SMA) (1:100; Abcam ab5694, Cambridge, United Kingdom), or myosin light chain (phospho S20, 1:100, Abcam ab2480, Cambridge, United Kingdom), and the capillary basement membrane and pericytes were labelledwith isolectin B4-Alexa Fluor 647 (1:200, overnight; Molecular Probes, I32450, Thermo Fisher Scientific, Waltham, MA). Z-stacks of the cortex and outer medulla (frame size 640.17 × 640.17 µm) for cell counting were acquired confocally (Zeiss LSM 700, Oberkochen, Germany). Pericyte intersoma distance was calculated between pairs of pericytes on capillaries within the same imaging plane. Kidney damage was assessed using kidney injury molecule-1 (Kim-1) antibody (1:100, overnight; Novus Biologicals, NBP1-76701, Abingdon, United Kingdom). Red blood cells were labelled with antibody to glycophorin A (1:2000, AbCam ab9520, Cambridge, United Kingdom). Alexa Fluor conjugated secondary antibodies were added overnight (1:500; ThermoFisher, A31572, A31556, A31570, Waltham, MA).
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