Protein lysates from human lung microvascular endothelial cells (HLMVECs) were prepared in radioimmunoprecipitation assay (RIPA) buffer (Millipore Sigma) as previously published.4 , 42 In brief, cells were scraped off in RIPA buffer on ice, then incubated on shaker for 30 min. at 4°C, followed by centrifugration at 13,000g for 15 min. Supernatants were aliquoted and protein quantification was performed using BioRad Protein Assay DC‐2 kit (5000112). Gel electrophoresis and Western blot were prepared as published previously4 , 42 and the following antibodies were used for analysis: β‐actin (loading control, Millipore Sigma; A5441), vascular cell adhesion molecule‐1 (VCAM1, Abcam; ab1067777), α‐smooth muscle antigen (α‐SMA, Agilent; M085129), SM22α (Cell Signaling Technologies; #4047) and SNAIL1 (Cell Signaling Technologies, #3879). Data were analyzed using band intensity with background subtraction in Image Studio (BioRad). Data were normalized versus β‐actin (loading control) and expressed as n‐fold of control antibody.
Sm22α
Manufactured by Cell Signaling Technology
Sourced in United States
SM22α is a protein that is expressed in smooth muscle cells. It is commonly used as a marker for the identification and characterization of smooth muscle cells in various experimental and research applications.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Cell Signaling Technology (2 mentions)
Image studio, by Bio-Rad (1 mentions)
Snail1, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Vimentin, by Solarbio (1 mentions)
Vcam 1, by Cell Signaling Technology (1 mentions)
U73122, by Bio-Techne (1 mentions)
Sp600125, by Bio-Techne (1 mentions)
Pluronic f 127 powder, by Merck Group (1 mentions)
Antibodies against, by Cell Signaling Technology (1 mentions)
Sb202190, by Bio-Techne (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Bw723c86, by Bio-Techne (1 mentions)
Ly272015, by Bio-Techne (1 mentions)
α smooth muscle actin α sma, by Cell Signaling Technology (1 mentions)
Pd98059, by Bio-Techne (1 mentions)
Ly294002, by Bio-Techne (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
5 protocols using sm22α
1
Western Blot Analysis of Endothelial Cells
2
Western Blot Analysis of Cell Lysates
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At the endpoint of treatment, cultured cells were lysed with RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor. The supernatant was extracted for the measurement of total protein with a protein assay kit (BCA). Samples were dissociated with SDS-PAGE and then transferred to PVDF membranes and incubated with primary antibody for GAPDH [Kangchen Biotech (Shanghai, China), Cat# KC-5G4, RRID:AB_2493106], α-SMA [Cell Signaling Technology (Beverly, MA, USA), Cat# 19245, RRID:AB_2734735], SM22α (Cell Signaling Technology, Cat# 52011), Vimentin [Solarbio (Beijing, China), Cat# GB11192, RRID:AB_2814685], P65 Cell Signaling Technology Cat# 8242S), acetyal-P65 (Cell Signaling Technology Cat# 12629S), and VCAM-1 (Cell Signaling Technology Cat# 39036, RRID:AB_2799146) at 4 °C overnight. The membranes were washed 3 times and incubated for 1 h at room temperature with horseradish-peroxidase-(HRP)-conjugated secondary antibodies. Images were quantified using the digital gel image analysis system TANON 5500, and ImageJ Version 1.46r analysis software was used for protein expression analysis.
3
Cultured Aortic SMCs Drug Treatments
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Rat or mouse aortic SMCs were isolated and grown in DMEM supplemented with 10% fetal bovine serum and antibiotics. SMCs were starved with serum‐free DMEM for 24 hours before drug treatments. BW723C86, LY272015, SP600125, SB202190, PD98059, LY294002, and U73122 were from Tocris Bioscience (Bristol, UK). 5‐HT, platelet‐derived growth factor‐BB (PDGF‐BB), 5‐bromo‐2′‐deoxyuridine (BrdU), and Pluronic F‐127 powder were from Sigma‐Aldrich (St. Louis, MO). Rapamycin was from Cell Signaling Technology (Beverly, MA). Antibodies against mammalian target of Rapamycin (mTOR), phosphorylated mTOR (Ser2448), p70S6 kinase, phosphorylated p70S6 kinase (Ser389), α‐smooth muscle actin (α‐SMA), and SM22α were from Cell Signaling Technology. Antibody against 5‐HT2BR was from BD Bioscience Pharmingen (San Diego, CA). Antibodies against 5‐HT receptor 2A (5‐HT2AR) and vimentin were from Abcam (Cambridge, MA). Antibodies against BrdU and CD31 were from Santa Cruz Biotechnology (Dallas, TX). Antibody against Mac‐2 was from Celdarlane (Burlington, ON, Canada).
4
Tissue Culture and Antibody Reagents
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All tissue culture reagents were purchased from Thermo Fisher Scientific; unless mentioned, all chemicals were obtained from Sigma Aldrich. Primary antibodies were purchased from Abcam UK (TNAP, OPN, β-actin, α-SMA, SM22α, CBS, and CSE) or Cell Signalling Technology (Runx2). Secondary antibodies were from Jackson Im-munoResearch Europe.
5
Western Blot Analysis of NLRP3 Inflammasome
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Samples were homogenized in lysis buffer, and the supernatant was extracted for the measurement of total protein with a protein assay kit (BCA; Pierce, Santa Cruz, CA, USA). Equal amounts of total protein were separated by SDS-PAGE, and transferred to PVDF membranes in Trisglycine methanol buffer. The bands were visualized with Enhanced Chemiluminescence Detection Kit (Thermo Scientific, Rockford, IL, USA). The antibodies against NLRP3, ASC and procaspase-1 were purchased from Abcam (Cambridge, MA, USA). Antibodies against α-SMA, SM22α, OPN and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). IL-1β and PCNA were purchased from Protein Tech Group Inc. (Chicago, IL, USA).
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