After treatment, the cells were collected and lysed in RIPA buffer. The protein concentrations were measured by a Bradford protein assay kit (Sigma-Aldrich). Equal amounts of protein were subjected to SDS-PAGE and then transferred to PVDF membranes (Millipore, Boston, MA, USA). After blocking with 5% skimmed milk for 1 hour at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4°C. The following antibodies were used: caspase-3 (No. 14214), Bcl-2 (No. 4223), Bcl-xl (No. 2762), Mcl-1 (No. 94296), Bax (No. 2774), cytochrome c (No. 4272), Smac/DIABLO (No. 15108), p-mTOR (No. 5536) and mTOR (No. 2972) and were purchased from Cell Signalling Technology (Danvers, MA, USA). Bax (6A7) (No. ab5714), p-Akt (No. ab81283, Ser 473), Akt (No. ab8805), p-PI3K (No. ab182651, Y607), PI3K (No. ab191606) and GAPDH (No. ab8245) were obtained from Abcam (San Diego, CA, USA). The membrane was then incubated with the secondary antibody and visualized by ECL (No. 32106) (Thermo Fisher Scientific, Rockford, IL, USA). Purification of cytosolic fractions and Bax immunoprecipitation was performed as described by Yu et al 2016.13 (link)
P mtor no 5536
Manufactured by Cell Signaling Technology
Sourced in United States
P-mTOR (No. 5536) is an antibody product from Cell Signaling Technology. It is used to detect the phosphorylated form of the mammalian target of rapamycin (mTOR) protein. mTOR is a serine/threonine protein kinase that plays a central role in regulating cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Bradford protein assay kit, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Ab62557, by Abcam (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
P tsc2, by Cell Signaling Technology (1 mentions)
2 protocols using p mtor no 5536
1
Apoptosis Signaling Pathway Analysis
2
Sunitinib, autophagy, and apoptosis signaling
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Sunitinib, CQ and all fluorescent dyes were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The primary antibodies against Bcl-2 (sc-492), Bax (sc-70407) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Primary antibodies against Beclin-1 (ab62557), autophagy related 5/12 (Atg5/12; ab78073) were purchased from Abcam (Cambridge, MA, USA). Primary antibodies against light chain 3 (LC3) I/II (no. 4108), p53 (no. 9282), caspase-3 (no. 9662), caspase-9 (no. 9508), phosphorylation of histone H3 (no. 9711), phosphoinositide 3-kinase (PI3K; no. 4255), Akt (no. 4685), phosphorylated (p)-Akt (no. 4058), tuberous sclerosis complex 2 (TSC2; no. 4308), p-TSC2 (no. 3617), p-mechanistic target of rapamycin (p-mTOR; no. 5536) and mTOR (no. 4517), p70 ribosomal S6 kinase (p70S6K; no. 2708) and p-p70S6K (no. 9204) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
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