Protein lysates were collected from cells that treated by si‐ESCO2 or si‐con, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF; protease inhibitor) was added into RIPA buffer to extract protein successfully. Then, protein was separated in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Finally, the target proteins were probed using primary antibodies and secondary antibodies. All the antibodies including ESCO2 (ab86003), GAPDH (ab181602), AKT (ab179463), p‐AKT (ab38449), mTOR (ab134903), p‐mTOR (ab109268), PCNA (ab92552), and p53 (ab32389) were purchased from Abcam. The protein bands were developed by enhanced chemiluminescence reagent (Thermo Fisher Scientific), and QUANTITY ONE was applied to detect the gray values of proteins.
P mtor ab109268
Manufactured by Abcam
Sourced in United Kingdom
P-mTOR ab109268 is a primary antibody that specifically recognizes the phosphorylated form of the mammalian target of rapamycin (mTOR) protein. mTOR is a serine/threonine protein kinase that plays a crucial role in regulating cell growth, proliferation, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin sc 6260, by Santa Cruz Biotechnology (2 mentions)
E cadherin sc 8426, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Ab32389, by Abcam (1 mentions)
Quantity one, by Thermo Fisher Scientific (1 mentions)
Gapdh ab181602, by Abcam (1 mentions)
Bicinchoninic acid kit, by Boster Bio (1 mentions)
Enhanced chemiluminescent substrate kit, by Merck Group (1 mentions)
Efemp2 12004 1 ap, by Proteintech (1 mentions)
Twist 25465 1 ap, by Proteintech (1 mentions)
Emt antibody sampler kit 9782, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
N cadherin sc 7939, by Santa Cruz Biotechnology (1 mentions)
Slug ab27568, by Abcam (1 mentions)
Snail ab167609, by Abcam (1 mentions)
Twist ab50887, by Abcam (1 mentions)
Fibulin 3 sc 33722, by Santa Cruz Biotechnology (1 mentions)
N cadherin sc 59987, by Santa Cruz Biotechnology (1 mentions)
Erk 9102, by Cell Signaling Technology (1 mentions)
Bcl 2, by Abcam (1 mentions)
5 protocols using p mtor ab109268
1
Protein Expression Profiling by Western Blot
2
Protein Expression Analysis of EMT Markers
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Experimental cells were harvested and then mixed with RIPA:PMSF (100:1) to facilitate total protein extraction, and protein concentration was then determined using a bicinchoninic acid kit (BOSTER). Equal concentrations (40 μg) of total protein were then separated by electrophoresis on a 10% separation gel and transferred to a polyvinylidene fluoride membrane. This membrane was then blocked for 1 h using nonfat milk (5%) and incubated overnight with a working solution of the relevant primary antibodies (EFEMP2 12004-1-AP, STEAP1 20199-1-AP, STEAP2 20201-1-AP, Twist 25465-1-AP, Proteintech; STEAP3 PA5-115969, STEAP4 PA5-115971, Invitrogen™; EMT Antibody Sampler Kit #9782, Cell Signaling Technology; PI3K ab86714, p-PI3K ab182651, AKT ab8805, p-AKT 38449, mTOR ab32028, p-mTOR ab109268, Abcam) at 4°C. The membranes were then washed using Tris-buffered saline with Tween® 20 and incubated with the secondary antibody for 1 h at room temperature. Membranes were then washed again and visualized using an enhanced chemiluminescent substrate kit (Millipore) and Image J software.
3
Western Blot Analysis of EMT Markers
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Cells were lysed on ice in RIPA (radio-immunoprecipitation assay) buffer with 1 mM PMSF (phenylmethylsulfonyl fluoride). From the cell lysate, 40 μg of total protein was loaded into the wells of the SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel elec trophoresis) gel, along with a molecular weight marker. Electrophoresis was carried out for 1–2 h at 100 V, followed by transfer to PVDF (polyvinyl difluoride) membranes, which were then blocked with 5% BSA (bovine serum albumin). The membranes were then incubated overnight at 4°C with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, vimentin sc-6260, Santa Cruz; fibulin-4 ab125073, Snail ab167609, Slug ab27568, Twist ab50887, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT 38449, mTOR ab32028, p-mTOR ab109268; Abcam) at working dilutions of 1:1000. After washing the membranes thrice with TBST for 5 min each, the membranes were incubated with conjugated secondary antibody diluted to 1:1000, at room temperature for 1 h. Blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).
4
Western Blot Analysis of EMT Markers
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Target cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor (RIPA:PMSF = 100:1). After determining the protein concentration by the Bicinchoninic Acid (BCA) method, protein samples (40 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 h with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin (BSA). The membranes were then incubated on a shaking bed with primary antibodies (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab106077, Twist ab50887, Zeb 2 ab138222, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT ab38449, mTOR ab32028, p-mTOR ab109268, Abcam; p-ERK (Thr202/Tyr204) #4370, ERK #9102, Cell Signaling), at a 1:2,000 dilution overnight at 4 °C. The next day, the membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Finally, positive labeling of the proteins on the membranes was visualized by enhanced chemiluminescence (ECL) using a Bio-Rad ECL kit (Solarbio).
5
Western Blot Analysis of Apoptosis Markers
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Primers were ordered from Genewiz Company (Beijing, China). Antibodies, including anti-CENPF (Ag29688), anti-GAPDH (10494-1-AP, 1:5,000), and HRP sheep anti-rabbit/mouse (1:5,000) were ordered from PTG Company (Bellevue, WA, USA); Active-Caspase3 (#ab32042, 1:1,000), Bcl-2 (ab32124, 1:1,000), Bax (ab32503, 1:1,000), Cyclin D1 (ab134175, 1:1,000), p-AKT (ab38449, 1:1,000), and p-mTOR (ab109268, 1:1,000) were purchased from Abcam (Cambridge, United Kingdom).
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