Cellular or whole lung lysates were run on a 10% SDS-PAGE gel and transferred using the Trans-Blot Turbo Transfer System (Bio-Rad). Low fluorescence polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) were activated in methanol and blocked with EveryBlot Blocking Buffer (Bio-Rad) followed by incubation with the following primary antibodies: OGR1 (“GPR68” Invitrogen, Carlsbad, CA, USA), beta-tubulin (Abcam, Cambridge, MA, USA), α-SMA (Sigma-Aldrich, St. Louis, MO, USA), phospho-Smad2/total Smad2 (Abcam), and Col1A1 (Aviva Systems Biology, San Diego, CA, USA). Immunofluorescent secondary antibodies (StarBright520 for mouse and StarBright700 for rabbit, Bio-Rad) were diluted at 1:20k in 5% milk and TBST and membranes were incubated for one hour. Membranes were imaged with a Chemidoc MP fluorescent imager (Bio-Rad) and quantified with Image Lab (version 6.1, Bio-Rad).
Starbright 520
Manufactured by Bio-Rad
Starbright 520 is a fluorescent dye used for labeling and detection in life science applications. It has an excitation maximum of 499 nm and an emission maximum of 520 nm, making it compatible with common fluorescent detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Starbright 700, by Bio-Rad (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Chemidoc mp fluorescent imager, by Bio-Rad (1 mentions)
β tubulin, by Abcam (1 mentions)
α sma, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Everyblot blocking buffer, by Bio-Rad (1 mentions)
Blocking reagent, by Bio-Rad (1 mentions)
Chemidoc station, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Gtx109669, by GeneTex (1 mentions)
Irdye 800, by LI COR (1 mentions)
4 protocols using starbright 520
1
Western Blot Analysis of Lung Lysates
2
SDS-PAGE and Western Blotting Protocol
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Denaturing sodium dodecyl sulfate (SDS) was performed, as published.18 (link),55 (link) 5-20 μg protein per lane were separated on a 15% SDS polyacrylamide gels using Tris/glycine buffer using 130 V for 1.5-2 hours. Proteins were transferred onto nitrocellulose membranes using a Trans-Blot Turbo Transfer System (Biorad). Membranes were blocked with Biorad Blocking reagent, incubated with primary antibodies dissolved in 3% bovine serum albumin in Tris-buffered saline. followed by fluorescent labeled secondary antibodies (Starbright 520 and Starbright 700 from Biorad; dilution 1:10,000). Signals were detected using a ChemiDoc station from Biorad. Gel and immunoblot images were processed using Image Lab (Biorad) and Adobe Photoshop and Illustrator. Densitometry was determined using Fiji/Image J.
3
Western Blot Analysis of Cardiac Proteins
4
Plasmid Generation and Antibody Detection
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FLAG-tagged Tg in pcDNA3.1+/C-(K)-DYK plasmid was purchased from Genscript (Clone ID OHu20241). Site-directed mutagenesis was then performed to generate FT-G2341R, FT-L2284P, FT-C1264R, FT-A2234D, and untagged Tg plasmids (supplemental Table S1 ). Primary antibodies were acquired from commercial sources and used at the indicated dilutions in immunoblotting buffer (5% bovine serum albumin in Tris-buffered saline pH 7.5, 0.1% Tween-20, and 0.1% sodium azide). Mouse monoclonal antibodies were used for the detection of KDEL (1:1000, Enzo Life Sciences, ADI-SPA-827), M2 anti-FLAG (1:1000, Sigma Aldrich, F1804). Polyclonal rabbit antibodies were used to detect Calnexin (1:1000, GeneTex, GTX109669), protein disulfide isomerase family A member 4 (PDIA4) (1:1000, Proteintech, 14712-1-AP), DNAJC10 (1:500, Proteintech, 13101-1-AP), thyroglobulin (1:1000, Proteintech, 21714-1-AP), UGGT1 (1:1000, Proteintech, 14170-1-AP), STT3A (1:2000, Proteintech, 12034-1-AP), and STT3B (1:2000, Proteintech, 15323-1-AP). Secondary antibodies were obtained from commercial sources and used at the indicated dilutions in 5% milk in Tris-buffered saline pH 7.5, 0.1% Tween-20 (TBS-T): Goat anti-mouse Starbright700 (1:10,000, Bio-Rad,12004158), Goat anti-rabbit IRDye800 (1:10,000, LI-COR, 926-32211), and Goat anti-rabbit Starbright520 (1:10,000, Bio-Rad,12005869).
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