For siRNA treatment, cells (GM10253A and HybNeo3; 10–20% confluent) were transfected with 10 nM Silencer Select Pre-designed siRNA targeting EXOSC3 (Ambion, Life Technologies) using Lipofectamine RNAi MAX (ThermoFisher) 24 h after seeding and again 48 h later. After a further 48 h exosome knockdown was confirmed by western blotting and TTseq was performed. Silencer Select RNA sequence for EXOSC3 were GAGATATATTCAAAGTTGA, part number s83102. The control RNA was Stealth RNAi siRNA Negative Control (ThermoFisher). For western blotting cells were suspended in NuPAGE LDS sample buffer (ThermoFisher) with 10 mM DTT, incubated at 100 °C for 5 min and sonicated briefly. Protein samples were resolved on 12% bis-tris gels (ThermoFisher) and transferred to Immobilon-P PVDF 0.45 mm membrane (Merck Millipore) by wet transfer. Membranes were probed with anti-EXOSC3 antibody (Abcam, Ab156683; 1:1000) using standard techniques and detected by enhanced chemiluminescence.
Immobilon p pvdf 0.45 mm membrane
Manufactured by Merck Group
Immobilon-P PVDF 0.45 mm membrane is a polyvinylidene fluoride (PVDF) membrane designed for use in various laboratory applications. It has a pore size of 0.45 micrometers and is a commonly used material for protein transfer and immobilization in Western blotting and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Silencer select pre designed sirna targeting, by Thermo Fisher Scientific (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (3 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Ab156683, by Abcam (1 mentions)
3 protocols using immobilon p pvdf 0.45 mm membrane
1
Exosomal RNA Knockdown Using siRNA
2
Silencing EXOSC3 by siRNA in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For siRNA treatment, cells (GM10253A and HybNeo3; 10%-20% confluent) were transfected with 10 nM Silencer Select Pre-designed siRNA targeting EXOSC3 (Ambion, Life Technologies) using Lipofectamine RNAi MAX (ThermoFisher) 24 h after seeding and again 48 h later. After a further 48 h exosome knockdown was confirmed by western blotting and TTseq was performed. Silencer Select RNA sequence for EXOSC3 were GAGATATATTCAAAGTTGA, part number s83102. The control RNA was Stealth RNAi siRNA Negative Control (ThermoFisher). For western blotting cells were suspended in NuPAGE LDS sample buffer (ThermoFisher) with 10 mM DTT, incubated at 100°C for 5 min and sonicated briefly. Protein samples were resolved on 12% bis-tris gels (ThermoFisher) and transferred to Immobilon-P PVDF 0.45 mm membrane (Merck Millipore) by wet transfer. Membranes were probed with anti-EXOSC3 antibody (Abcam, Ab156683) using standard techniques and detected by enhanced chemiluminescence.
3
Silencing EXOSC3 by siRNA in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For siRNA treatment, cells (GM10253A and HybNeo3; 10%-20% confluent) were transfected with 10 nM Silencer Select Pre-designed siRNA targeting EXOSC3 (Ambion, Life Technologies) using Lipofectamine RNAi MAX (ThermoFisher) 24 h after seeding and again 48 h later. After a further 48 h exosome knockdown was confirmed by western blotting and TTseq was performed. Silencer Select RNA sequence for EXOSC3 were GAGATATATTCAAAGTTGA, part number s83102. The control RNA was Stealth RNAi siRNA Negative Control (ThermoFisher). For western blotting cells were suspended in NuPAGE LDS sample buffer (ThermoFisher) with 10 mM DTT, incubated at 100°C for 5 min and sonicated briefly. Protein samples were resolved on 12% bis-tris gels (ThermoFisher) and transferred to Immobilon-P PVDF 0.45 mm membrane (Merck Millipore) by wet transfer. Membranes were probed with anti-EXOSC3 antibody (Abcam, Ab156683) using standard techniques and detected by enhanced chemiluminescence.
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