The Arp2/3 complex inhibitor CK-66633 (link) was purchased from Tocris Bioscience and dissolved in DMSO. BSA, β-Lactoglobulin and Pluronic F127 were purchased from Sigma and dissolved in 1x PBS. pLL-PEG was from Surface SolutionS and diluted in 10~mM HEPES. GFP-FAK was provided by Ekaterina Papusheva/ Carl-Philipp Heisenberg. Speckle GFP-Vinculin was a gift from Alan Rick Horwitz. MRLC-GFP was a gift from Rex Chisholm. siRNAs were Stealth Select RNAi siRNAs from Invitrogen with the following sequences: TLN1 siRNA (RSS320275): 5'-GGGCAUAUCCAUGUCUUCGAGCAAA-3'; TLN2 siRNA (RSS321623): 5'-GAGAGGAGCCGAGAAGCGAAUAUUU-3'. Knockdown efficiency was quantified by Western blotting with antibodies for TLN1 (monoclonal, clone 8d4, Sigma #T3287, 1:2000), TLN2 (GeneTex, #EPR2536(2), 1:1000) and GAPDH (clone 1D4, NovusBiologicals #NB300-221, 1:20000). Representative images of immunoblots are based on at least 2 experiments.
Protocol detail
Arp2/3 Complex Inhibitor CK-66633 Protocol
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Antibodies
Arp2 3 complex
Clone
Dmso
Gapdh
Hepes
Immunoblots
Pluronic f127
Rnai
Sirna
Vinculin
β lactoglobulin
Corresponding Organization :
Other organizations : University College London, MRC Laboratory for Molecular Cell Biology, Max Planck Institute for the Physics of Complex Systems
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Variable analysis
independent variables
- CK-66633 (Arp2/3 complex inhibitor) concentration
- SiRNA targeting TLN1 and TLN2
- Knockdown efficiency of TLN1 and TLN2 proteins, quantified by Western blotting
- DMSO (solvent for CK-66633)
- 1x PBS (solvent for BSA, β-Lactoglobulin, and Pluronic F127)
- 10 mM HEPES (solvent for pLL-PEG)
- Antibodies used for Western blotting (anti-TLN1, anti-TLN2, and anti-GAPDH)
- Positive control: None mentioned
- Negative control: None mentioned
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