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.
Image studio
Manufactured by Bio-Rad
Image Studio is a software platform designed for the acquisition, analysis, and visualization of digital images generated from Bio-Rad's imaging systems. The software provides a user-friendly interface for managing and processing data from various imaging techniques, including western blots, gels, and blots.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Sm22α, by Cell Signaling Technology (1 mentions)
Snail1, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
3 protocols using image studio
1
Western Blot Analysis of Endothelial Cells
2
Western Blot Analysis of Apoptosis Markers
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The RIPA lysis buffer (Beyotime, Shanghai, China) was used to lyse the cell pellets and then collected the supernatants and determined the protein concentrations by BCA protein assay kit (Beyotime, Shanghai, China). The protein extracts were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, United States), and incubated with specific antibodies of OXR1 (Proteintech, 13514-1-AP), p53 (Proteintech, 10442-1-AP), p21 (Proteintech, 28248-1-AP), Bax (Proteintech, 50599-2-Ig), cleaved caspase-3 (Proteintech, 19677-1-AP), and GAPDH (Proteintech, 10494-1-AP) at dilution of 1:500 incubated at 4°C overnight after being blocked with 5% non-fat dry milk in Tris-buffered saline containing 1% Tween-20 (TBST) at room temperature for 2 h. Then, the membranes were incubated with secondary antibody at room temperature for 2 h and exposed to the enhanced chemiluminescence-plus reagents according to the manufacturer’s protocol after extensive washing. Finally, the protein bands were detected using Bio-Rad Image Studio and normalized against GAPDH.
3
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The RIPA lysis buffer (Beyotime, Shanghai, China) was used to lyse the cell pellets and then collected the supernatants and determined the protein concentrations by BCA protein assay kit (Beyotime, Shanghai, China). The protein extracts were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, United States), and incubated with specific antibodies of OXR1 (Proteintech, 13514-1-AP), p53 (Proteintech, 10442-1-AP), p21 (Proteintech, 28248-1-AP), Bax (Proteintech, 50599-2-Ig), cleaved caspase-3 (Proteintech, 19677-1-AP), and GAPDH (Proteintech, 10494-1-AP) at dilution of 1:500 incubated at 4°C overnight after being blocked with 5% non-fat dry milk in Tris-buffered saline containing 1% Tween-20 (TBST) at room temperature for 2 h. Then, the membranes were incubated with secondary antibody at room temperature for 2 h and exposed to the enhanced chemiluminescence-plus reagents according to the manufacturer’s protocol after extensive washing. Finally, the protein bands were detected using Bio-Rad Image Studio and normalized against GAPDH.
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