HUVECs were lysed in NP40 lysis buffer (Boston Bioproducts) including cOmplete™ Mini EDTA‐free Protease Inhibitor Cocktail (Roche; Sigma‐Aldrich) and PhosSTOP™ Phosphatase Inhibitor tablets (Roche; Sigma‐Aldrich). The lysed supernatant was obtained by centrifugation at 4°C for 10 min at 13,000 rpm and the total protein content was quantified by a Micro BCA Protein assay kit (Thermo Fisher Scientific). The proteins were boiled in Laemmli SDS sample buffer (Alfa Aesar) for 10 min and separated by Mini‐Protean TGX Gels 4%−20% (Bio‐Rad), transferred onto an Immobilon‐PSQ PVDF Membrane (EMD Millipore). Membranes were probed with antibodies against β‐arrestin1/2, phospho‐eNOS (Ser1177), total‐eNOS, Myc‐tag, Erk5, GAPDH (CST, 1:1000), IFT81, IFT74 (Proteintech; 1:1000), GFP (Sigma‐Aldrich), and BMPR‐II (BD Bioscience; 1:1000). Bound primary antibodies were detected by secondary anti‐rabbit or anti‐mouse IgG‐HRP (CST, 1:2000). Blots were developed with Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).
Ift74
Manufactured by Proteintech
IFT74 is a protein purified from insect cells that is a component of the intraflagellar transport complex B. It plays a role in the assembly and maintenance of cilia and flagella.
Automatically generated - may contain errors
Lab products found in correlation
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx gels 4 20, by Bio-Rad (2 mentions)
Immobilon psq pvdf membrane, by Merck Group (2 mentions)
Phosstop phosphatase inhibitor tablet, by Roche (2 mentions)
Laemmli sds sample buffer, by Thermo Fisher Scientific (2 mentions)
Np 40 lysis buffer, by Boston BioProducts (2 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (2 mentions)
Bmprii, by BD (2 mentions)
Ift81, by Proteintech (1 mentions)
2 protocols using ift74
1
Immunoblotting of Endothelial Cell Lysates
2
HUVECs Protein Extraction and Western Blotting
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HUVECs were lysed in NP40 lysis buffer (Boston Bioproducts) including cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche; Sigma-Aldrich) and PhosSTOP™ Phosphatase Inhibitor tablets (Roche;
Sigma-Aldrich). The lysed supernatant was obtained by centrifugation at 4℃ for 10 min at 13,000 rpm and the total protein content was quantified by a Micro BCA Protein assay kit (Thermo Scientific). The proteins were boiled in Laemmli SDS sample buffer (Alfa Aesar) for 10 min and separated by Mini-Protean TGX Gels 4-20 % (Bio-Rad), transferred onto an Immobilon-PSQ PVDF Membrane (EMD Millipore).
Membranes were probed with antibodies against β-arrestin1/2, phospho-eNOS (Ser1177), total-eNOS, Myc-tag, Erk5, GAPDH (CST, 1:1000), IFT81, IFT74 (Proteintech, 1:1000), GFP (Sigma-Aldrich) and BMPR-II (BD Bioscience, 1:1000). Bound primary antibodies were detected by secondary anti-rabbit or anti-mouse IgG-HRP (CST, 1:2000). Blots were developed with Supersignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
Sigma-Aldrich). The lysed supernatant was obtained by centrifugation at 4℃ for 10 min at 13,000 rpm and the total protein content was quantified by a Micro BCA Protein assay kit (Thermo Scientific). The proteins were boiled in Laemmli SDS sample buffer (Alfa Aesar) for 10 min and separated by Mini-Protean TGX Gels 4-20 % (Bio-Rad), transferred onto an Immobilon-PSQ PVDF Membrane (EMD Millipore).
Membranes were probed with antibodies against β-arrestin1/2, phospho-eNOS (Ser1177), total-eNOS, Myc-tag, Erk5, GAPDH (CST, 1:1000), IFT81, IFT74 (Proteintech, 1:1000), GFP (Sigma-Aldrich) and BMPR-II (BD Bioscience, 1:1000). Bound primary antibodies were detected by secondary anti-rabbit or anti-mouse IgG-HRP (CST, 1:2000). Blots were developed with Supersignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
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