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2 protocols using phospho akt s473 4060

1

Western Blot Analysis of Protein Expression

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Proteins were extracted using RIPA lysate (Beyotime) supplemented with 1% PMSF (Beyotime) and 2% phosphatase inhibitor (Beyotime). Following electrophoretic separation through SDS-PAGE, the proteins were transferred onto PVDF membranes. After blocking with 5% skim milk, primary antibodies specific for FOXC1 (ab227977, Abcam,1:1000), CD44 (A19020, Abclonal,1:1000), CD133 (A0219, Abclonal,1:1000), CBX7 (ab178411, Abcam,1:1000), IGF-1R (ab182408, Abcam,1:1000), phosphor-IGF-1R (ab39398, Abcam,1:1000), Akt (4691, Cell Signaling Technology,1:1000), phospho-Akt (S473) (4060, Cell Signaling Technology,1:1000), ERK1/2 (ab184699, Abcam,1:1000), phospho -ERK1/2 (ab201015, Abcam,1:1000) primary antibodies overnight and β-actin (AC026, Abclonal,1:10000) as internal reference were used for protein examination. .
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2

Detailed Antibody Procurement Protocol

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Mouse anti‐Akt1 (#2967), anti‐ubiquitin (#3936), anti‐Myc (#2276), rabbit anti‐BAP1 (#13271), anti‐PTEN (#9559), and phospho‐Akt‐S473 (#4060) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti‐BAP1 (#10398‐1‐AP) and mouse anti‐beta‐actin (#60008‐1‐Ig) antibodies were purchased from Protein Technology (Rosemont, IL, USA). Mouse anti‐PTEN (ab170941) antibody was purchased from Abcam (Cambridge, UK). Mouse anti‐beta Tubulin (100109‐MM05T) antibody was purchased from Sino Biological (Beijing, China). Anti‐Flag M2 (F1804) antibody was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Anti‐HA (16B12) antibody was purchased from Covance (Berkeley, CA, USA). Anti‐GST (#M0807‐1) antibody was purchased from HuaBio (Hangzhou, Zhejiang, China). Protein G Plus/A agarose suspension (#IP05) was purchased from Calbiochem (San Diego, CA, USA).
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