Tissue or cells were lysed in 2x Laemmli buffer and boiled for 5 min at 100 °C followed by centrifugation at 20,000 × g for 15 min at 4 °C. The protein extracts were subjected to standard Western blot analysis which was performed. The following antibodies were used for western blotting: Rabbit polyclonal antibody against CCM3 was generated (Invitrogen) against full-length recombinant human CCM3 protein expressed and purified from Escherichia coli. β-actin (mouse, A1978) and β-actin (mouse, A5441) were from Sigma; p-Caveolin-1 (rabbit, 611339) was from BD Pharmingen; Abl (rabbit, 2862s), p-Akt (rabbit, 9271), Akt (rabbit, 9272), p-MLC2 (rabbit, 3674), PDGFR-β (rabbit, 3168), p-Tie2 (rabbit, 4221), p-Tie2 (rabbit, 4226), Tie2 (rabbit, 4224), and VEGFR2 (rabbit, 2479) were from Cell Signaling Technology. Caveolin-1 (rabbit, sc894) was from Santa Cruz. Angpt2 (rabbit, ab8452), p-Abl (rabbit, ab4717), and N-Cadherin (rabbit, ab76057) were from Abcam; Angpt2 (AF7186) was from R&D Systems. All primary antibodies were diluted 1:1000. For data presented in the same figure panel, the samples were derived from the same experiment and that gels/blots were processed in parallel. Uncropped blots and gel images were provided in the Source data file.
P mlc2
Manufactured by Cell Signaling Technology
Sourced in United States
P-MLC2 is a laboratory reagent used in cell biology research. It is an antibody that detects phosphorylated myosin light chain 2, a protein involved in cell motility and contractility. The antibody can be used to monitor the activation state of this protein in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Tenascin c, by Abcam (4 mentions)
Total fak, by BD (4 mentions)
Py397 fak, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Lsm 780 confocal microscope, by Zeiss (4 mentions)
β actin, by Merck Group (3 mentions)
α sma, by Merck Group (3 mentions)
Rock1, by Cell Signaling Technology (3 mentions)
Fibronectin, by Abcam (2 mentions)
Gli 1, by Thermo Fisher Scientific (2 mentions)
Collagen 12a1, by Santa Cruz Biotechnology (2 mentions)
Integrin β1, by Merck Group (2 mentions)
Mabt409, by Thermo Fisher Scientific (2 mentions)
P mypt1, by Merck Group (2 mentions)
Collagen 3, by Abcam (2 mentions)
Total stat3, by Cell Signaling Technology (2 mentions)
Rock2, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
P smad2 3, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
25 protocols using p mlc2
1
Western Blot Analysis of CCM3 Signaling
2
Immunohistochemistry Antibody Panel
3
Antibody Validation for Cellular Analysis
4
Western Blot and Nuclear Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as previously described10 (link). Nuclear/cytoplasm separation was performed using an NE-PER kit (Pierce). The following antibodies have been used: NF-κBp65 (Millipore, 17–10,060; 1:1,000), EFNB2 (GeneTex, GTX88049; 1:1,000), VEGFA (SantaCruz Biotechnology, sc-152; 1:2,000), MLC2 (Cell Signalling, 3672; 1:1,000), p-MLC2 (Ser19; Cell Signalling, 3675; 1:500), MYPT1 (Cell Signalling, 8574; 1:1,000), p-MYPT1 (Cell Signalling, 4563; 1:500), A/C Laminin (Active Motif, 39287; 1:1,000) and Tubulin (Cell Signalling, 2148; 1:1,000).
5
Cardiomyocyte Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cardiomyocytes were collected and lysed in RIPA lysis buffer (Beyotime). The protein concentration was determined using a BCA Protein Assay kit (Beyotime). All of the proteins were separated by 10% SDS‐polyacrylamide gels (Invitrogen) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), followed by blocking in 5% skim milk in TBST (tris, buffer, solution, tween) for 1 hour at room temperature. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MLCK (1:1000 dilution, bs‐9865R; Bioss, China), MLC2 (1:2000 dilution, F109.3E1; Enzo Life Science, Farmingdale, USA), p‐MLC2 (1:1000 dilution, #3671; Cell Signaling Technology, MA, USA), cleaved caspase‐3 (1:500 dilutions, NB100‐56113; Novus Biologicals, USA), Bax (1:2000 dilution, #2772, Cell Signaling Technology), Bcl‐2 (1:2000 dilution, ab59348; Abcam, MA, USA) and GAPDH (1:10000 dilution, ab37168; Abcam). Next, the membranes were incubated with the appropriate horseradish peroxidase‐conjugated secondary antibody for 1 hour at 37°C. The proteins were scanned and detected using a FluorChem E System (Cell Biosciences), and ImageJ software was used to determine the intensity of each protein band.
6
Immunofluorescence Staining of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the cells were grown on the micropatterned substrates for 3 or 7 days, they were gently washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature, and permeabilised with 0.2% Triton X-100 in PBS for 10 minutes at room temperature. The cells were blocked with 5% bovine serum albumin (Boster, Wuhan, China) for 30 minutes, incubated with goat anti-mouse polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) against alkaline phosphatase (ALP, Cat# sc-365765), type I collagen (COL I, Cat# sc-59772), or osteocalcin (OCN, Cat# sc-390877) at a 1:100 dilution for 60 minutes at 37°C, washed with PBS three times, and incubated in the dark for 60 minutes at room temperature with a fluorescein isothiocyanate-labelled affinity-purified antibody to goat IgG (Invitrogen) at a 1:1000 dilution. The staining method used for YAP (A1002, ABclonal, Woburn, MA, USA) and tetraethyl rhodamine isothiocyanate-labelled P-MLC2 (Cat # 3674, Cell Signaling Technology, Boston, MA, USA) was the same as described above. Fluorescence images of these proteins were analysed by ImageJ software. The nuclear/cytoplasmic ratio of YAP in MSCs and MC3T3-E1 cells was obtained by dividing the intensity value in the nucleus to that in the cytoplasm.
7
Molecular Mechanisms of Epithelial Barrier
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In our research, SDC, GYY4137, FITC-dextran (4 KDa, FD-4), and FITC-dextran (40 KDa, FD-40) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies for immunofluorescence and western blotting were purchased from companies as follows: Occludin (Thermo Fisher Scientific, Waltham, MA, USA); MLCK (Abcam, Cambridge, UK); P-MLCK (Abcam, Cambridge, UK); ZO-1 (Cell Signaling Technology, Danvers, MA, USA); MLC2 (Cell Signaling Technology, Danvers, MA, USA); and P-MLC2 (Cell Signaling Technology, Danvers, MA, USA).
8
Immunofluorescence Staining Protocol for Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise indicated, immunofluorescence (IF) was performed as described previously (Durgan et al., 2015 (link)). Briefly, cells were fixed using 3.7% formaldehyde in PBS (10 min, RT), permeablised in 0.5% triton (5 min, RT) and then incubated with primary antibody in PBS (4C, overnight): β-catenin (BD 610153; RRID:AB_397554 ; 1:100), p-MLC2 (Cell Signalling 3671L; RRID:AB_330249 ; 1:100), LC3 (Cell Signalling 4108; RRID:AB_2137703 ; 1:100), LAMP1 (BD 555798; 1:100), p-Histone H3 (Millipore 06–570; RRID:AB_310177 ; 1:100), ZO-1 (Invitrogen 61–7300; RRID:AB_2533938 ; 1:100). Cells were washed in PBS and incubated with Alexa Fluor 488/568 goat anti-mouse/rabbit (H+L) secondary (1:500) and Hoechst 3342 (1μg/ml) for 45 min at RT; where indicated, HCS CellMASK Deep Red (Thermofisher, H32721) or Alexa Fluor 488-phalloidin (Cell Signalling, 8878S) were included, to stain the cell body or actin respectively, according to the manufacturers’ guidelines. Cells were washed with PBS, then water, and mounted using Prolong Gold Antifade Mountant (Thermofisher). Image acquisition was performed with a Confocal Zeiss LSM 780 microscope (Carl Zeiss Ltd) equipped with a 40X oil immersion 1.4 NA objective, using Zen software (Carl Zeiss Ltd).
9
Immunohistochemistry Antibody Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were as follows: a-SMA (Sigma-Aldrich C6198, 1:1000), GLi-1 (Thermo Scientific PA5-32206, 1:500), Tenascin C (Abcam AB108930, 1:500), Fibronectin (Abcam AB2413, 1:500), Collagen 12A1 (Santa Cruz Biotechnology E-15 sc-68449, 1:200), Sox2 (Abcam ab97959, 1:500), Vimentin (Cell Signaling 5741S, 1:200), FAP (Abcam AB53066, 1:500), Collagen III (Abcam AB7778, 1:500), YAP (Cell signaling 4912, 1:200), β1integrin (EMD Millipore MABT409, 1:500), pY397-FAK (Invitrogen 44625, 1:200), total FAK (BD Biosciences 610088, 1:1,000), ROCK1 (Cell Signaling C8F7, 1:1,000), ROCK2 (Cell Signaling D1B1, 1:1,000), p-MLC2 (Cell Signaling 3671, 1:200), p-MyPT1 (Millipore ABS45, 1:200), p-Stat3 (Cell Signaling 9145, 1:200), p-SMAD2/3 (Cell Signaling 8828, 1:200), total Stat3 (Cell Signaling 9132, 1:1,000), GAPDH (Cell Signaling 2118, 1:5,000), CD45 (BD Biosciences 550539, 1:200), CD68 (Thermo Scientific Ab-3, 1:200), Alexa Fluor-conjugated goat secondary anti–mouse IgG and anti–rabbit IgG antibodies (Invitrogen A11012, 1:1,000 and A11005, 1:1,000) and HRP-conjugated rabbit secondary antibody (GE health care life sciences NA934VS, 1:5,000).
10
Tissue Immunofluorescence Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were fixed in 4% PFA overnight and paraffin-processed. We cut 5-µm sections from paraffin-embedded blocks for H&E staining and immunohistochemistry. Adherent cells cultured in two-well chamber slides were stained by immunocytochemistry.
The following antibodies were used for immunofluorescence at the indicated concentrations: DDR1 (1:50; Santa Cruz Biotechnology, sc-532), E-cadherin (1:100; BD Biosciences, 610181), K8 (1:50; Developmental Studies Hybridoma Bank, TROMA-I), K14 (1:5000 [Convance, PRB-155P] and 1:100 [Santa Cruz Biotechnology, sc-17104]), α-SMA Cy3 conjugate (1:250; Sigma, C6198), vimentin (1:200; Sigma, V5255), DDR2 (1:50; LifeSpan BioSciences, LS-C164363), phH3 (1:100; Cell Signaling, 9701), HIF1α (1:50; Novus Biologicals, NB100-479), pMLC2 (1:100; Cell Signaling Technology, 3671), goat anti-mouse IgM μ chain Cy3 conjugate (1:200; Jackson ImmunoResearch, 115-166-075), and Alexa 488 anti-rat, Alexa 488 anti-rabbit, Alexa 488 anti-mouse, Alexa 568 anti-rabbit, and Alexa 647 anti-goat secondary antibodies (1:500; Molecular Probes, A11006, A24922, A24920, A21069, and A21447). Nuclei were stained with DAPI (Vector Laboratories, H-1200). Confocal or fluorescence microscopy was performed on a Nikon C1si confocal microscope or a Keyence BZ-X700 fluorescence microscope.
The following antibodies were used for immunofluorescence at the indicated concentrations: DDR1 (1:50; Santa Cruz Biotechnology, sc-532), E-cadherin (1:100; BD Biosciences, 610181), K8 (1:50; Developmental Studies Hybridoma Bank, TROMA-I), K14 (1:5000 [Convance, PRB-155P] and 1:100 [Santa Cruz Biotechnology, sc-17104]), α-SMA Cy3 conjugate (1:250; Sigma, C6198), vimentin (1:200; Sigma, V5255), DDR2 (1:50; LifeSpan BioSciences, LS-C164363), phH3 (1:100; Cell Signaling, 9701), HIF1α (1:50; Novus Biologicals, NB100-479), pMLC2 (1:100; Cell Signaling Technology, 3671), goat anti-mouse IgM μ chain Cy3 conjugate (1:200; Jackson ImmunoResearch, 115-166-075), and Alexa 488 anti-rat, Alexa 488 anti-rabbit, Alexa 488 anti-mouse, Alexa 568 anti-rabbit, and Alexa 647 anti-goat secondary antibodies (1:500; Molecular Probes, A11006, A24922, A24920, A21069, and A21447). Nuclei were stained with DAPI (Vector Laboratories, H-1200). Confocal or fluorescence microscopy was performed on a Nikon C1si confocal microscope or a Keyence BZ-X700 fluorescence microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!