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4 protocols using anti p akt 4060s

1

Protein Expression Analysis by Western Blot

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Protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were shifted onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membrane was incubated with the primary antibodies at 4°C overnight. The primary antibodies are listed as below: anti-hexokinase 2 (anti-HK-2; 2867S; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-cleaved caspase 3 (9661S; Cell Signaling Technology), anti-matrix metallopeptidase 2 (anti-MMP2; 40994S; Cell Signaling Technology), anti-cyclin D1 (55506S; Cell Signaling Technology), anti-TPX2 (12245S; Cell Signaling Technology), anti-phosphoinositide-3-kinase (anti-PI3K; 4257S; Cell Signaling Technology), anti-AKT (14982S; Cell Signaling Technology), anti-phosphorylated-AKT (anti-p-AKT; 4060S; Cell Signaling Technology), and anti-β-actin (ab8226; Abcam, Cambridge, MA, USA). Subsequently, the membrane was incubated with the secondary antibody for 1 h at room temperature. Protein bands were visualized using the imaging system (Thmoregan Biological Technology, Beijing, China). Western blot assay was carried out as previously described [21 (link)].
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2

Western Blot Assay for S100A11

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The antibodies that we used for the Western Blot assay are as follows: Primary antibody: anti-S100A11 (10237-1-AP) was purchased from Proteintech (USA), anti-GAPDH (P04406), anti-E-cadherin(#14472), anti-ERK (#4695T), anti-p-ERK (#4370T), anti-AKT (#5691S) and anti-p-AKT (#4060S) were from Cell Signaling Technology (Shanghai, China), anti-N-cadherin (ab76011) was from Abcam company (MA, United States); Secondary antibodies: anti-mouse IgG, HRP-linked antibody(#7076) and mouse anti-rabbit IgG mAb (HRP conjugate) were also purchased from Cell Signaling Technology (Shanghai, China). PrimScript RT reagent Kit with gDNA Eraser (RR047A) was from Takara (Japan), and SYBR Green PCR Master Mix was from Thermo Fisher Scientific (USA).
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3

Western Blot Analysis of EGFR, HER2, and AKT Signaling

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Cell were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Equal amounts of proteins from cells were separated by 4%–12% SDS-PAGE and were transferred to a PVDF membrane. The blots were blocked for 1 h with 5% Bovine Serum Albumin and then incubated over night with the following primary antibodies: Anti-EGFR (2256, Cell Signaling), Anti-pEGFR (2234, Cell Signaling), anti-HER2 (SC52349, Santa Cruz), anti-pHER2 (2247, Cell Signaling), anti-HER3 (SC81455, Santa Cruz), anti-pHER3 (4791, Cell Signaling), anti-AKT (2920, Cell Signaling), anti-pAKT (4060S, Cell Signaling) anti-P70S6K (49D7, Cell Signaling), anti-pP70S6K (108D2, Cell Signaling), and anti-GAPDH (ab9485, Abcam). Membranes were incubated with their respective secondary antibodies for 1h and analyzed using the enhanced chemiluminescence (ECL) system. Antibody detection and quantification were conducted using the iBright ™ FL1000 (ThermoFisher) and the iBright analysis software.
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4

Western Blot Analysis of Protein Expression

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Total proteins were extracted from tissue samples using RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitors (Sigma Aldrich, St. Louis, MO, USA). Total proteins were then separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were blocked using 5% BSA in TBST buffer and incubated with the following primary antibodies: mouse anti-E-cadherin (610181A) and anti-N-cadherin (610,920, BD Biosciences, San Jose, CA, USA), rabbit anti-PD-L1 (13684S), anti-AKT (9272S), and anti–p-AKT (4060S) (Cell Signaling Technology, Houston, TX, USA), mouse anti-β-actin (sc-47778) and anti-mouse (sc-2005). Membranes were then washed and incubated with the appropriate secondary antibody conjugated with HRP: anti-mouse (sc-2005) and anti–rabbit (sc-2357, Santa Cruz Biotechnology). Specific protein bands were visualized using the ECL reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA). Protein expression levels were normalized against β-actin levels. All experiments were performed in triplicate.
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