After cell harvest and tissue collection, total proteins were extracted via radioimmunoprecipitation assay (RIPA; Thermo Fisher Scientific, Waltham, MA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate proteins from the loading products to the gels; then, proteins on the gels were transferred to the polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). PVDF membranes were blocked in 5% skim milk (Invitrogen), followed by the incubation of primary antibodies against cyclin D1 (#2922, 1:1000), p21 (#2947, 1:1000), p27 (#3686, 1:1000), B-cell lymphoma-2 (Bcl-2; #4223, 1:1000), Bcl-2-associated X (Bax; #5023, 1:1000), phospho-AKT (p-AKT; #4060, 1:2000), AKT (#4691, 1:1000), phospho-mTOR (p-mTOR; #5536, 1:1000), mTOR (#2983, 1:1000) or internal control glyceraldehyde-phosphate dehydrogenase (GAPDH; #8884, 1:1000) at 4°C overnight. Enhanced chemiluminescence (ECL; Bio-Rad) was exploited for protein presentation after the membranes were incubated with a second antibody (#7074, 1:3000) for 1 h at room temperature. The protein bands were captured and analyzed by Image Lab software (Bio-Rad). The antibodies used in this study were purchased from Cell Signaling Technology (CST, Boston, MA, USA).
P mtor
Manufactured by Thermo Fisher Scientific
The P-mTOR is a laboratory equipment product that measures the phosphorylation of the mammalian target of rapamycin (mTOR) protein. mTOR is a serine/threonine protein kinase that regulates cell growth, proliferation, and survival. The P-mTOR equipment provides a quantitative assessment of mTOR phosphorylation levels, which is a key indicator of mTOR pathway activity.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Thermo Fisher Scientific (3 mentions)
P pi3k, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Skim milk, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Chemiluminescence system, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Pro caspase 1, by Abcam (1 mentions)
Cleaved caspase 1, by Abcam (1 mentions)
Unc5b, by Abcam (1 mentions)
As1012, by Aspen (1 mentions)
Pro il 1β, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Nf κb p65, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Bio Basic (1 mentions)
4 protocols using p mtor
1
Protein Expression Analysis via RIPA-SDS-PAGE
2
Western Blot Analysis of Signaling Proteins
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Western blot was performed as previously described [20 (link)]. Firstly, protein samples were separated and shifted onto PVDF membranes (Millipore, Billerica, MA). Subsequent to sealing with nonfat milk, the membranes were subjected to overnight incubation at 4°C with the following primary antibodies against IQGAP3 (1.7 mg/mL, Invitrogen), CDCA5 (0.5 mg/mL, Invitrogen), EGFR (1 mg/mL, Sigma-Aldrich), K-Ras (1 mg/mL, Thermo Fisher Scientific), p-B-Raf (1 mg/mL, Sigma-Aldrich), p-MEK (~1 mg/mL, Invitrogen), MEK (~1 mg/mL, Invitrogen), p-ERK (0.5 mg/mL, Invitrogen), ERK (0.5 mg/mL, Invitrogen), p-mTOR (0.5 mg/mL, Invitrogen), mTOR (1 mg/mL, Thermo Fisher Scientific), p-PI3K (1 mg/mL, Invitrogen), PI3K (1 mg/mL, Thermo Fisher Scientific), p-AKT (1 mg/mL, Invitrogen), AKT (0.1 mg/mL, Invitrogen), and GAPDH (0.5 mg/mL, Merck, Darmstadt, Germany). Then, the blots went through incubation with secondary antibody (~2 mg/mL, Sigma-Aldrich). At length, chemiluminescence system supplied by GE Healthcare (Chicago) was utilized for protein detection. The experiment was performed in triplicate.
3
Protein Extraction and Immunoblotting Protocol
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Total protein was extracted from cells or tissues using a whole protein extraction kit (AS1012, ASPEN). The primary antibodies (ASPEN) used were netrin-1 (1:1000), CD63 (1:2000), CD9 (1:2000), TSG101 (1:1000), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) (1:1000), apoptosis-associated speck-like protein (ASC) (1:1000), pro-caspase-1 (1:2000), cleaved caspase-1 (1:2000), pro-IL-1β (1:2000), IL-1β (1:2000) (Abcam), Unc5b (1:2000), PI3K (1:2000), p-PI3K (1:2000), Akt (1:2000), p-Akt (1:2000), mTOR (1:2000), p-mTOR (1:2000) and GAPDH (1:2000) (26,616, Thermo). A gel image processing system (gel Pro analyser software) was used to analyse the grey value of the target strip.
4
Protein Detection in Brain Tissues
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RIPA lysis buffer and Bradford Protein Assay Kit were used for protein determination from brain tissues (Bio Basic Inc., Markham, Ontario, Canada). The separated proteins were loaded and incubated on the PVDF membrane with primary antibodies for Beclin 1 (# PA5-20172), p-ERK (#44-680G), p-mTOR (#44-1125G), p-PI3K (# PA5-99367), ERK ( #13–6200), mTOR (# AHO1232), LC3 A (#PA5-23180), LC3 B (#PA1-16930), NF‐κB (p65) (# 51–0500), and β-actin (# MA5-15739) (Thermo Fisher Scientific Inc., Waltham. MA, USA) at 4°C. Following washing, blots were incubated with peroxidase‐labeled anti‐rabbit secondary antibodies at 37°C for 1 h. protein Bands intensity was detected using the chemiluminescent substrate (ClarityTM Western ECL substrate—Bio-Rad, TNC, USA), and a CCD camera-based imager. Analysing the images was done using Chemi Doc MP Imager and then normalised by β-actin.
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