Cells were lysed in RIPA buffer (Thermo Fisher) including the following protease and phosphatase inhibitors; Complete Mini, EDTA free (Roche), Protease Inhibitor cocktail (Sigma), Halt™ Phosphatase Inhibitor cocktail (Thermo Fischer) and phosphatase inhibitor cocktails 2 (Sigma) and 3 (Sigma). Proteins were separated in SDS-PAGE and transferred onto nitrocellulose membrane (Thermo Fisher). Membranes were blocked for 1 hour with 5% dry milk in TBS containing 0.05% Tween-20 at room temperature. Membranes were probed with primary antibodies overnight at 4° C and proteins were visualized with ECL detection kit (GE Healthcare) as previously described (16 (link)). ERβ was immunoblotted with two rabbit polyclonal (Millipore, GeneTex) and the 14C8 antibody (GeneTex) and recombinant ERβ protein (Thermo Fisher) was loaded to ensure specificity. Antibodies against RhoC, ERα, pAKT1, AKT1, GST, ELMO1, ROCK1, HER3 and GAPDH were from Cell signaling. Anti-β-Actin antibody was from Abcam and anti-GPR141 was from Thermo Fisher. All antibodies are listed in Supplementary Table 2 .
Protocol detail
Western Blot Analysis of Protein Targets
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RIPA buffer
Complete Mini
Protease Inhibitor cocktail
Halt™ Phosphatase Inhibitor cocktail
phosphatase inhibitor cocktails 2
nitrocellulose membrane
ECL detection kit
Anti-β-Actin antibody
Actin
Akt1
Anti antibody
Antibodies
Antibody
Buffer
Cells
Edta
Gapdh
Her3
Inhibitors
Membranes
Milk
Nitrocellulose
Phosphatase
Phosphatase inhibitor 2
Protease
Protease inhibitor
Proteins
Rabbit
Recombinant protein
Ripa
Rock1
Sds page
Tween 20
Corresponding Organization : University of Pennsylvania
Other organizations : Cedars-Sinai Medical Center, The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Lysis of cells in RIPA buffer
- Addition of protease and phosphatase inhibitors (Complete Mini, EDTA free, Protease Inhibitor cocktail, Halt™ Phosphatase Inhibitor cocktail, phosphatase inhibitor cocktails 2 and 3)
- Protein expression levels of ERβ, RhoC, ERα, pAKT1, AKT1, GST, ELMO1, ROCK1, HER3, GAPDH, β-Actin, and GPR141
- Separation of proteins using SDS-PAGE
- Transfer of proteins onto nitrocellulose membrane
- Blocking of membranes for 1 hour with 5% dry milk in TBS containing 0.05% Tween-20 at room temperature
- Probing of membranes with primary antibodies overnight at 4°C
- Visualization of proteins using ECL detection kit
- Recombinant ERβ protein (Thermo Fisher) loaded to ensure specificity of ERβ antibodies
- Not explicitly mentioned
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