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3 protocols using beta actin n 21

1

Quantitative Immunoblotting of PSMC2, PSMD2, and ERα

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For PSMC2 and PSMD2 immunoblotting, cells were lysed in HENG buffer (50mM Hepes-KOH pH 7.9, 150mM NaCl, 2mM EDTA pH 8.0, 20mM sodium molybdate, 0.5% Triton X-100, 5% glycerol), with protease inhibitor cocktail (Roche Diagnostics #11836153001). Protein concentration was determined by the BCA assay (Thermo-Fisher Scientific #23227), and proteins were resolved on SDS-PAGE for immunoblot analysis. Antibodies against the following human proteins were used: alpha-Tubulin (ab80779; Abcam), PSMC2 (MSS1–104; Enzo Life Sciences) and PSMD1 (C-7; Santa-Cruz). Visualization was performed using the ChemiDoc MP System (Bio-Rad), and ImageLab Software (Bio-Rad) was used to quantify relative band intensities. For ERα immunonblotting, cells were lysed with a mix of 4X protein loading buffer (Li-Cor 928–40004) and 10X NuPAGE sample reducing agent (Life Technologies NP0009). Protein concentration was normalized by cell counting, and proteins were resolved on SDS-PAGE. Antibodies against the following human proteins were used: beta-Actin (N-21; Santa Cruz), ERα (F-10; Santa Cruz). Visualization was performed using the Odyssey CLx imaging machine (Li-Cor), and Image Studio Software (Li-Cor) was used to quantify the relative intensities.
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2

Quantitative Immunoblotting of PSMC2, PSMD2, and ERα

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For PSMC2 and PSMD2 immunoblotting, cells were lysed in HENG buffer (50mM Hepes-KOH pH 7.9, 150mM NaCl, 2mM EDTA pH 8.0, 20mM sodium molybdate, 0.5% Triton X-100, 5% glycerol), with protease inhibitor cocktail (Roche Diagnostics #11836153001). Protein concentration was determined by the BCA assay (Thermo-Fisher Scientific #23227), and proteins were resolved on SDS-PAGE for immunoblot analysis. Antibodies against the following human proteins were used: alpha-Tubulin (ab80779; Abcam), PSMC2 (MSS1–104; Enzo Life Sciences) and PSMD1 (C-7; Santa-Cruz). Visualization was performed using the ChemiDoc MP System (Bio-Rad), and ImageLab Software (Bio-Rad) was used to quantify relative band intensities. For ERα immunonblotting, cells were lysed with a mix of 4X protein loading buffer (Li-Cor 928–40004) and 10X NuPAGE sample reducing agent (Life Technologies NP0009). Protein concentration was normalized by cell counting, and proteins were resolved on SDS-PAGE. Antibodies against the following human proteins were used: beta-Actin (N-21; Santa Cruz), ERα (F-10; Santa Cruz). Visualization was performed using the Odyssey CLx imaging machine (Li-Cor), and Image Studio Software (Li-Cor) was used to quantify the relative intensities.
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3

Investigating AUY922 and Imatinib Effects

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The-AUY922 compound was kindly provided by Novartis institution for BioMedical Research. Imatinib was purchased from LC laboratories. Imatinib was diluted in PBS and AUY922 was dissolved in 100% DMSO (dimethyl sulfoxide) at the stocking concentration of 10 mM, and was aliquoted into microtubes to be stored at −20°C until use. Commercially available antibodies used were anti-phospho-Jak2Y1007 (Mollipore, Cat# 04-1098), Stat3 (Cell signaling, Cat# 9132), Akt (Cell signaling, Cat# 9272), HSP90 (Cell signaling, Cat# 4874), c-Abl (Cell Signaling, Cat# 2862), phosphotyrosine (4G10) (Millipore, Cat# 05-321), alpha tubulin (B-7) (Santa Cruz Biotechnology, Cat# sc-5286), beta actin (N-21) (Santa Cruz Biotechnology, Cat# sc-130656). Sepharose beads conjugated Jak2 antibody was purchase from Cell Signaling (Cat# 4089). Anti-Abl SH2 domain monoclonal antibody (8E9) was produced by our own lab.
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