The ability of the newly synthesised compound RM37 to dissociate the native MDM2–p53 complex was assessed by a quantitative immuno-enzymatic assay, as previously reported [19 (link)]. Briefly, lysate samples of GBM cells were incubated for 10 min at room temperature with the solvent DMSO (control) (Sigma-Aldrich, Milan, Italy) or with increasing concentrations of the compound RM37 (from 1 nM to 50 µM) before being transferred to the wells coated with the antibody anti-MDM2 (sc-965, Santa Cruz Biotechnology, 1550 in 0.05% poly-L-ornithine) (Sigma-Aldrich, Milan, Italy). Following washings to remove unbound MDM2 and BSA treatment to block nonspecific sites, each sample was incubated for 90 min with the anti-p53 antibody (sc-6243, Santa Cruz Biotechnology, 15250 in 5% milk). The levels of the MDM2–p53 complex were quantified using an HRP-conjugated antibody and the TMB substrate.
Sc 965
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-965 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed to perform specific functions within the research and testing environment. The core function of the Sc-965 is to enable users to carry out their research and analysis objectives, though a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Sc 126, by Santa Cruz Biotechnology (2 mentions)
T5168, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Sc 47778, by Santa Cruz Biotechnology (2 mentions)
Yo pro 1, by Thermo Fisher Scientific (2 mentions)
Ab13847, by Abcam (2 mentions)
Myoseverin, by Merck Group (2 mentions)
4 4 diisothiocyanatostilbene 2 2 disulphonic acid, by Thermo Fisher Scientific (2 mentions)
Flivo tracer, by ImmunoChemistry Technologies (2 mentions)
Ab6673, by Abcam (2 mentions)
Staurosporine, by BD (2 mentions)
Z vad, by Santa Cruz Biotechnology (2 mentions)
Am4300, by Thermo Fisher Scientific (2 mentions)
Q vd oph, by Santa Cruz Biotechnology (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Sc 6243, by Santa Cruz Biotechnology (1 mentions)
Dmso control, by Merck Group (1 mentions)
Ab109520, by Abcam (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Immobilon p, by Merck Group (1 mentions)
19 protocols using sc 965
1
Quantifying MDM2-p53 Complex Dissociation
2
Western Blot Analysis of Drug-Treated Cells
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After treating the cells with a concentration of the drug similar to the GIC90 value, cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM sodium vanadate, 1 mM EDTA, 50 mM NaF, 1% Triton X-100, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) with sonication at 4 °C. Protein lysate was resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred to a polyvinylidene difluoride membrane filter (Immobilon P; Millipore, Burlington, MA, USA). The blots were subjected to immunoblotting analysis using primary antibodies against the following: MDM2 (1:500 dilution; sc-965, SANTA CRUZ BIOTECHNOLOGY, INC, Santa Cruz, CA, USA), RPL11 (1:1000 dilution; 3A4A7, Invitrogen, Waltham, MA, USA), p53 (1:400 dilution; sc-126, SANTA CRUZ BIOTECHNOLOGY, Inc., Dallas, TX, USA), P21 (1:3000 dilution; ab109520, Abcam, Cambridge, UK), and Actin (1:3000 dilution; PM053, MEDICAL & BIOLOGICAL LABORATORIES Co., LTD, Tokyo, Japan). Immunoreactive band signal intensities were detected using LuminoGraph II (ATTO, Tokyo, Japan).
3
Co-immunoprecipitation of HDAC1 and MDM2
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Co-IP experiment was performed according to the manufacturer’s instructions of Pierce Co-IP kit (Thermo Scientific, #26149). NPCs were harvested and homogenized in ice-cold lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol; pH 7.4) with 2× complete protease inhibitor cocktails (Roche). Nuclei and debris were pelleted at 10,000 × g for 20 min. The supernatant was collected and the protein concentrated was quantified. The resulting supernatant was pre-cleared for 1 h with Pierce Control Agarose Resin. A monoclonal antibody against HDAC1 (BioVersion, 3601-30) or MDM2 (Santa Cruz, sc-965) was incubated with AminoLink Plus coupling Resin for 2 h and washed 3 times with coupling buffer (10 mM sodium phosphate, 150 Mm NaCl; pH 7.2). Meanwhile, a monoclonal antibody against IgG (Cell signaling, #5415s) was utilized as a negative control. The pre-cleared lysates (1 mg of proteins) were immunoprecipitated with antibody-coated Resin at 4 °C for 2 h. After three washes with lysis buffer, the precipitates were then separated on an SDS-PAGE for protein analysis and probed with anti-MDM2 or anti-HDAC1 antibody, respectively.
4
Antibody Validation for Western Blot and Immunofluorescence
5
Western Blot Analysis of MDM2 Isoforms
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Cells were harvested with Trypsin EDTA (Lonza) and lysed with IPH buffer with protease inhibitor, debris were removed, and proteins were denatured by boiling in SDS buffer and loaded on 12% SDS-PAG (Bio Rad) with PS11 protein ladder (GeneOn). Separated proteins were transferred onto 0.2-μM nitrocellulose membranes by turbo blotting for 7 minutes, 2.5A, and 25 V using the Bio Rad system. Unspecific protein binding was blocked by incubation in 5% nonfat milk in TBS-Tween0.05% for 1 hour at room temperature or overnight at 4°C. Membranes were subsequently incubated in MDM2 specific antibody Sc-813 (Santa Cruz) recognizing exon 3. Potentially expressed proteins from MDM2-Δ5 were investigated using different antibodies having their epitope in exon 3 (Sc 813, Santa Cruz), exon 8 (Sc-965, Santa Cruz), and exon 12 (OP 146, Merck Millipore), respectively. GAPDH (SantaCruz) was used as loading control. Following washing of membranes in TBS-Tween0.05%, proteins bound by the primary antibody were detected by HRP-conjugated secondary antibody (Sigma). Signals were detected using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and the LAS 4000 imager (GE Healthcare).
6
Protein Interaction and Truncation Analyses
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Full-length OTUD3 WT, full-length OTUD3 C76A, full-length GRP78, and full-length PTEN were cloned into the pCMV-Myc, pFlag-CMV-2 or pEGFP-C1 vectors as indicated. GST-tagged OTUD3, GST-tagged OTU + UBA (1–340 aa), UBA (184–340 aa), Tail (341–398 aa) truncations were cloned into the pGEX-4T-2 vector. G1 (1–124 aa), G2 (125–399 aa), G3 (281–499 aa), G4 (500–654 aa) truncations of GRP78 were cloned into the pEGFP-C1 vector. Antibodies used in immunoblotting were: anti-actin (1:1,000, sc-1616, Santa Cruz); GAPDH (1:1,000, sc-25778, Santa Cruz);anti-GRP78 (1:1000; 11587-1-AP, Proteintech); anti-OTUD3 (1:500; HPA028543, Sigma); anti-PTEN (1:1000; #9188, Cell Signaling) and anti-pSer473-AKT (1:1000; #4060, CellSignaling); Anti-Flag (1:1000; sc-965, Santa Cruz); anti-Myc (1:1000; sc-374171, Santa Cruz); anti-HA (1:1000; M180-3, MBL); anti-GFP (1:1000; 66002-1-Ig, Proteintech).Normal IgG (sc-2003, Santa Cruz).
7
Western Blot Analysis of Cell Signaling
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Cells were washed with phosphate-buffered saline (PBS) and lysed with tissue lysis buffer (20 mM Tris-base, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM sodium orthovanadate, 1 mM benzamidine, and 1 mM phenylmethysulfonyl fluoride). Total cell extracts were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), transferred to Immobilon-P membranes (Millipore, Bedford, USA), and blotted with antibodies against Ell3 (ab67415, Abcam, Cambridge, USA), p53 (#2524, Cell Signaling, Denver, USA), Caspase-9 (#9665, Cell Signaling), NQO1 (sc-16464, Santa Cruz Biotechnology, Dallas, USA), phospho-ERK (#4370, Cell Signaling), total ERK (sc-145, Santa Cruz Biotechnology), lamin B (sc-6216, Santa Cruz Biotechnology), ɤH2AX (ab22551, Abcam), MDM2 (sc-965, Santa Cruz Biotechnology) and β-actin (sc-47778, Santa Cruz Biotechnology). Immunoreactivity was detected by enhanced chemiluminescence (ECL; Bio-Rad, CA, USA).
8
Western Blotting for Brain Protein Analysis
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For western blotting, mice were anesthetized, decapitated and brains were removed quickly. Crude protein was extracted by homogenizing 0.2 mg cerebral cortex in 200 µl RIPA lysis buffer containing 2 µl 1× complete protease inhibitor mixture. Lysates were centrifuged at 12,000 rpm at 4 °C for 10 min, then the supernatants were transferred to new tubes added with 1× loading buffer, and heated for 5 min at 95 °C. Protein was separated by SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane at 200 mA for 2 h in 4 °C. The membrane was blocked for 2 h in 5% non-fat dry milk and then incubated with primary antibodies: rabbit anti-ARGLU1 (1:500; HPA034962, Sigma-Aldrich), rabbit anti-p21 (1:1000; cell signaling, 2947 T), rabbit anti-PUMA (1:1000; Abcam, ab9643), rabbit anti-NOXA (1:1000; Abcam, ab131088), rabbit anti-p53 (1:1000; LSBio, LS-C334397), mouse anti-p53 (1;1000; Abcam ab26), mouse anti-MDM2 (1:200; Santa Cruz sc-965), mouse anti-MDM4 (1:200; Santa Cruz sc-74468), and anti-β-actin (1:5000; Millipore), followed by incubation with HRP-conjugated secondary antibody. ECL detection kit (Bio-Rad) was used for signal detection. Data were analyzed using ImageJ (NIH)-Fuji software.
9
Multimarker Immunofluorescence Staining
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The antibody used in immunofluorescence staining were as follows: anti-DDX4 (ab13840, Abcam), anti-GATA4 (ab124265 Abcam), anti-c-KIT (25-1171-82, eBioscience) anti-SYCP3 (ab97672, Abcam), anti-STRA8 (ab49602, Abcam), anti-γH2AX (ab2893, Abcam), anti-H3pSer10 (ab5176, Abcam), anti-MPS1 (ab11108, Abcam), anti-H2BK120ubi (#5546, CST), anti-MDM2 (sc-965, Santa Cruz), anti H2B (ab1790, Abcam), and anti-ACTIN (sc-47778, Santa Cruz). All fluorophore-conjugated secondary antibodies were obtained from Jackson Immuno Research.
10
Co-Immunoprecipitation of MDM2 Protein
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