The whole cell lysates or subcellular fractions were boiled with 5 × sodium dodecyl sulfate (SDS) sample buffer for 10 min at 100 °C and subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated in blocking buffer (5% skim milk) for 1 h and incubated overnight at 4 °C with primary antibodies against SIRT1 (1:1000; Cell Signaling Technologies, Beverly, MA, USA), p53 (1:1000; Cell Signaling Technologies), acetyl-p53 (Lys382) (1:1000; Cell Signaling Technologies), vimentin (1:1000; Cell Signaling Technologies), β-actin (1:2000; Cell Signaling Technologies), lamin B (1:4000; ProteinTech), CK-18 (1:2000; ProteinTech), CK-19 (1:2000; ProteinTech), and β-tubulin (1:2000, Sungene Biotech, Tianjin, China). Then, the membranes were washed and incubated at room temperature for 1 h with the appropriate horseradish peroxidase-linked secondary antibodies (1:4000; Abbkine, Redlands, CA, USA). The protein bands were visualized using Pierce™ ECL Western blotting substrate (Merck Millipore).
Acetyl p53 lys382
Manufactured by Cell Signaling Technology
Sourced in United States
Acetyl-p53 (Lys382) is a lab equipment product from Cell Signaling Technology. It is an antibody that specifically detects p53 protein acetylated at lysine 382. This modification is involved in the regulation of p53 activity.
Automatically generated - may contain errors
Lab products found in correlation
Sirt1, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Lamin b, by Proteintech (1 mentions)
Pierce ecl western blotting substrate, by Merck Group (1 mentions)
β tubulin, by Sungene Biotech (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Rapamycin, by Promega (1 mentions)
Z vad fmk, by Promega (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Gfp trap ma beads, by Proteintech (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Amersham nitrocellulose membranes, by GE Healthcare (1 mentions)
Gapdh 14c10, by Cell Signaling Technology (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
X ray film, by Fujifilm (1 mentions)
6 protocols using acetyl p53 lys382
1
Immunoblotting Analysis of Cellular Proteins
2
Adenovirus Protein Expression Assay
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Antibodies to Daxx, LC3A/B, p53 and acetyl-p53 (Lys382) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to E1A and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc, Dallas, TX, USA).
3-MA, rapamycin and pan caspase Inhibitor Z-VAD-FMK were purchased from Promega (Madison, WI, USA). Anti-adenovirus type 5 (Ad5) antibody was purchased from Abcam (Abcam, Cambridge, UK). Antibodies to IVa2, L4-22K, and L4-33K were kindly provided by Dr. Patrick Hearing at SUNY Stony Brook. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Trizol was purchased from Life Technologies (Carlsbad, CA, USA), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3-MA, rapamycin and pan caspase Inhibitor Z-VAD-FMK were purchased from Promega (Madison, WI, USA). Anti-adenovirus type 5 (Ad5) antibody was purchased from Abcam (Abcam, Cambridge, UK). Antibodies to IVa2, L4-22K, and L4-33K were kindly provided by Dr. Patrick Hearing at SUNY Stony Brook. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Trizol was purchased from Life Technologies (Carlsbad, CA, USA), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Immunoprecipitation of Acetylated p53
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Equal numbers of sorted cells were resuspended in lysis buffer (25 mM Tris-HCL [pH=7.5], 137 mM NaCl, 1 mM EDTA, 1% NP40, 1 mM DTT, 10% Glycerol) supplemented with 1 × CLAP protease-inhibitor cocktail (Chymostatin, Leupeptin, Antipain and Pepstatin A, 1 μg/ml each), 0.1 mM PMSF and 5 mM sodiumbutyrate. IP targeting endogenous protein: Cell lysates were incubated for 4 h at 4 °C with 2 μg antibody. Immunoprecipitation was carried out using protein G beads (Dynabeads; Invitrogen, Landsmeer, the Netherlands) overnight at 4 °C. IP targeting GFP-fusion proteins: Cell lysates were incubated with 25 μl GFP-trap_MA beads (Chromotek, Planegg-Martinsried, Germany) overnight at 4 °C. Immune complexes were washed three times with lysis buffer, boiled in SDS lysis buffer and further analyzed by immunoblotting as described above. Primary antibodies for Co-IP: acetyl-p53 (Lys382; Cell Signaling Technology; catalog number 2525L).
4
Western Blot Analysis of p53 Signaling Pathway
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Cell lysates were harvested by 2% SDS lysis buffer, heated and sonicated. Equal quantities of protein were loaded onto and separated by SDS–polyacrylamide gels (4–20% Mini-PROTEAN TGX Gel, BioRad, Watford, UK). The separated proteins were transferred and immobilised onto Amersham nitrocellulose membranes (GE Healthcare Life Science, Amersham, UK). Primary antibodies against p53 (DO-7) (M7001, Dako, Glostrup, Denmark), MDM2 (Ab-1) (OP46, Merck Millipore, Watford, UK), p21WAF1 (EA10) (OP64, Calbiochem, Merck Millipore), WIP1 (F-10) (sc-376257, Santa Cruz Biotechnology, Dallas, TX, USA), phospho-p53(Ser-15) (9284, Cell Signaling Technology, Danvers, MA, USA), acetyl-p53(Lys382) (2525, Cell Signaling Technology), BAX (2772, Cell Signaling Technology), GAPDH (14C10) (2118, Cell Signaling Technology) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (P0447/P0448, Dako) were used. All antibodies were diluted in 5% (w/v) non-fat milk or BSA in TBS–Tween. Proteins were visualised using enhanced chemiluminescence (GE Healthcare Life Sciences) and X-ray film (Fujifilm, Bedford, UK). Densitometry was carried out using ImageJ software (National Institutes of Health, Rockville, MD, USA).
5
Regulation of HDAC and p53 in Lung and Colorectal Cancers
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A549, H1299 and H460 human lung cancer cells purchased from the American Type Culture Collection (Manassas, VA, USA), Lu99 human lung cancer cells, purchased from the RIKEN cell bank (Tsukuba, Japan), and HCT 116 colorectal cancer cells (p53 null and p53 wild) were supplied by Dr. Kee-Ho Lee (KIRAMS, KOREA) were grown in the recommended growth medium (Invitrogen, Carlsbad, CA, USA). SAHA was purchased from ALEXIS Corporation (Lausen, Switzerland). Antibodies against HDAC1, HDAC2, HDAC3, cIAP2, Mdm2, HA, Myc and β-actin were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HDAC4, SIRT1, SIRT2, histone 3, acetyl-histone 3, acetyl-histone 4, acetyl-p53 (Lys382), puma, ubiquitin, caspase 3, cleaved PARP, p-ATM, ATM, p-ATR, ATR, p-Chk1, Chk1, p-Chk2, Chk2, p-γH2AX, γH2AX and survivin antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). XIAP, caspase 7 and p21 antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA, USA), and the p53 antibody was from Novocastra Lab. Ltd. (Newcastle, UK). The Flag antibody, Nutlin-3A and MG132 were from Sigma. (St Louis, MO, USA). The siRNAs targeting HDAC1, HDAC2, HDAC3, or HDAC4 were from Santa Cruz Biotechnology. Two different HDAC2 siRNAs (siHDAC #2 and siHDAC #3) and p53-specific siRNA were purchased from Ambion (Austin, TX, USA).
6
Protein Expression Analysis by Western Blot
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For each experimental condition and from each sample total, protein extracts were obtained, as described in [19 (link)]. For the analysis, 20 μg of each sample were loaded on Tris–glycine gradient gels (4% to 15% gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and separated at 100 V. To probe proteins with specific primary antibodies antibodies, they were transferred to PVDF membranes (Biorad Laboratories, Inc., Hercules, CA, USA). All the secondary antibodies used were horseradish peroxidase conjugated. All the antibodies were used as indicated by manufacturer. The following primary antibodies were used for Western blot: PARP (Cell Signaling, #9542), BCL-2 (Abcam, ab182858), BAX (Santa Cruz Biotechnology, sc-493), Acetyl-p53 Lys382 (Cell Signaling, #9542). As the internal control we used β-Actin (Cell Signaling, #3700), which was used as the loading control. To detect protein levels, Clarity western ECL (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used. The quantization was then obtained using ImageJ software vJ1, an open source tool. For each experimental condition a triplicate was performed, and results are expressed as the mean ± SD.
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