Western blot analysis was carried out as previously described [63 (link)]. Primary antibodies used in these studies were: cleaved PARP, cleaved caspase-3, phospho-histone H2A.X (Ser139), PLK1, PSMB5 and c-Myc (Cell Signaling Technology, Danvers, MA), Mcl-1 (BD Biosciences, San Jose, CA), α-tubulin (EMD Millipore, Billerica, MA), actin (Sigma-Aldrich, St. Louis, MO), NOXA (Enzo Life Sciences, Farmingdale, NY).
Psmb5
Manufactured by Cell Signaling Technology
Sourced in United States
PSMB5 is a proteasome subunit that is a key component of the 20S proteasome core complex. The 20S proteasome is responsible for the degradation of ubiquitinated proteins in the cell.
Automatically generated - may contain errors
Lab products found in correlation
Doxorubicin, by Merck Group (2 mentions)
Psmb8, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Psmb6, by Cell Signaling Technology (2 mentions)
Psmb7, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Phospho histone h2a x ser139, by Cell Signaling Technology (1 mentions)
Mcl 1, by BD (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by American Type Culture Collection (1 mentions)
Vascular cell basal medium, by American Type Culture Collection (1 mentions)
U ch2, by American Type Culture Collection (1 mentions)
U ch1, by American Type Culture Collection (1 mentions)
L glutamine, by American Type Culture Collection (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Endothelial cell growth kit bbe, by American Type Culture Collection (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
7 protocols using psmb5
1
Western Blot Analysis of Apoptosis Markers
2
Chordoma Cell Line Cultivation and Compound Screening
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Hoechst 33342 stains were purchased from Life Technologies (Cat no. H3570). Human Chordoma cell lines U-CH1 (Cat. no. CRL-3217) and U-CH2 (Cat. no. CRL-3218) were purchased from ATCC (Manassas, VA, USA). Cells were grown in Iscove’s Modified Dulbecco’s Medium (Cat. no. 30-2005, ATCC): RPMI-1640 Medium (Cat. no. 30-2001, ATCC) (4:1), 10% FBS (Cat. no. 30-2020, ATCC) supplemented with 2 mM L-glutamine (Cat. no. 30-2214, ATCC). HEK-293 (Cat. no. CRL-1573) and HUVEC (Cat. no. PCS-100-013) cells were purchased from ATCC and were grown in Eagle’s Minimum Essential Medium (Cat. no. 30-2003, ATCC) supplemented with 10% fetal bovine serum, and Vascular Cell Basal Medium (Cat. no. PCS-100-030, ATCC) supplemented with Endothelial Cell Growth Kit-BBE (Cat. no. PCS-100-040, ATCC), respectively. PSMB5 (Cat. no. 11903), PSMB8 (Cat. no. 13635), and β-Actin (Cat. no. 3700) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Doxorubicin was purchased from Sigma (Cat. no. D1515). Compound libraries were provided by the Institute of Chemistry and Cell Biology (ICCB) Longwood, Harvard Medical School, Boston, MA, USA.
3
Protein Extraction and Immunoblotting
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Cells were lysed by adding Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 5% Glycerol, 1% SDS)–containing protease and phosphatase inhibitors (Roche). The protein quantification was carried out by detergent-compatible protein assay kit (Bio-Rad, Hercules, CA), and 30 μg of protein was resolved in 12% SDS-PAGE gel. The antibodies used in this study are NFATC2, PSMB5, VIM (all from Cell Signaling), ENO3 (Sigma), FOXO1 (Immunoway), SMYD3 (Abcam), FOXP3 (eBioscience), and β-actin (CalBioChem).
4
Molecular Mechanisms of NRF2-p62 Axis
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Antibodies recognizing SOX2, KLF4, NANOG, MRP2, BCRP, CUL3, p62, LC3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and PSMB5 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against PSMB6 and PSMB7 were purchased from Enzo Life Sciences (Farmingdale, NJ, USA). NRF2, KEAP1, Ub, lamin B, and β-tubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The luciferase reporter plasmid containing the ARE was a gift from Dr. Wakabayashi (University of Pittsburg, PA, USA). The lentiviral expression plasmids for human NRF2 or p62 short hairpin RNA (shRNA), MissionTM Lentiviral Packaging Mix, hexadimethrine bromide, puromycin, 5-FU, doxorubicin, CHX, and MTT were from Sigma-Aldrich (Saint Louis, MO, USA). PI was purchased from Biolegend (San Diego, CA, USA). Hoechst 33342 (H342) and carboxy-H2DCFDA were purchased from Life Technologies (Carlsbad, CA, USA). The SYBR premix ExTaq system was obtained from Takara (Otsu, Japan). Substrates for proteasome proteases (Suc-LLVY-AMC, Z-LLE-AMC, and Z-ARR-AMC) were from EMD Chemicals (Darmstadt, Germany).
5
Western Blot Protein Analysis
6
Immunoblot Analysis of Proteasome Subunits
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Cells were trypsinized, centrifuged, washed, and then lysed in Hepes (100 mM; pH 7.5), EDTA (2 mM), NaF (100 mM), sodium chloride (500 mM), and trypsin inhibitor (50 μg/ml; Promega) with fresh cOmplete Mini Protease Inhibitor (Roche). Protein lysates (5–20 μg) were resolved by SDS–PAGE and probed overnight at 4°C with antibodies against PSMB5 (#12919), PSMB6 (#13267), PSMB7 (#13207), PSMB8 (#13726), and PA28g (#2142S) from Cell Signaling, used at 1:1,000, or β-tubulin (SAB4700544, 1:200; Merck). Chemiluminescent detection was carried out using appropriate secondary antibodies conjugated to horseradish peroxidase and the enhanced chemiluminescence kit (Amersham).
7
Protein Expression Analysis in Drug-Treated Cells
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Cells were cultured in the given concentration of drug/s for 4h, and then lysed in 10mM Tris/HCl buffer (pH 7.4) containing 137mM NaCl, 10% Glycerol, 1% NP40, 10mM β-glycerophosphate, 2mM sodium vanadate, 2mM NaF, 10mM sodium pyrophosphate, and cOmpleteTM EDTA-free protease inhibitor (Sigma-Aldrich). Lysates were subjected to SDS-PAGE with pre-cast 4-20% TGX gels (ThermoFisher), proteins transferred to nitrocellulose membranes, which were blocked for at least 1h at room temperature in either 5% skim milk powder in 50mM Tris/HCl buffer (pH 7.4) containing 150mM NaCl and 1% Tween 20 if imaging on a LAS-4000 (GE Healthcare), or Li-Cor PBS Odyssey Blocking Buffer if imaging on an Odyssey (Li-Cor, Lincoln, NE). Primary antibodies used were ATF4, XBP1, PSMB5, PSMB6, PSMB7 (Cell Signaling Technology), and β-actin (Sigma-Aldrich). Secondary antibodies used were HRP-conjugated goat-anti-rabbit and HRP-conjugated goat-anti-mouse (ThermoFisher), and goat-anti-rabbit IR and goat-anti-mouse IR 680RD (Li-Cor).
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