Mouse anti-VE-cadherin antibody (sc-9989) diluted 1:50 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (Philadelphia, PA, USA) and Molecular Probes (Eugene, OR, USA), respectively, and used for immunocytochemistry. HIF2α inhibitor [51 (link)] (Axon 2034) was obtained from Axon Medchem (Hanzeplein, The Netherlands). Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, Irvine, CA, USA) and Santa Cruz Biotechnology (sc-3067, Dallas, TX, USA). Marimastat from Santa Cruz Biotechnology (sc-202223, Dallas, TX, USA), MG-132 from Promega (G9951, Madison, WI, USA). Cytotoxgreen was obtained from Essen BioScience (Ann Arbor, MI, USA) and CellROXgreen from Molecular Probes (Eugene, OR, USA). PX-ethyl was purchased from Sigma (St. Louis, MO, USA). According to the Sigma safety data sheet, safety measures of eye shields, face shields, full-face respirator, and gloves should be taken. PX was used according to our previously reported protocol [43 (link),44 (link)] All other reagents applied in this study were used in accordance to the known literature and the supplier’s guidelines.
Mg 132
Manufactured by Promega
Sourced in United States
MG-132 is a proteasome inhibitor that blocks the activity of the 26S proteasome, a complex responsible for the degradation of ubiquitinated proteins in cells. It is a widely used tool in cell biology and biochemistry research.
Automatically generated - may contain errors
Lab products found in correlation
Marimastat, by Santa Cruz Biotechnology (2 mentions)
Cellrox green, by Thermo Fisher Scientific (2 mentions)
Cytotox green, by Sartorius (2 mentions)
Px ethyl, by Merck Group (2 mentions)
Cy and alexa fluor conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Z vad fmk, by Adooq (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Luciferase reporter constructs, by Promega (1 mentions)
Tubulin, by Cell Signaling Technology (1 mentions)
Sb203580, by Promega (1 mentions)
Ly294002, by Promega (1 mentions)
Pn3 sp1 fl, by Addgene (1 mentions)
Pn3 sp3 fl, by Addgene (1 mentions)
P erk, by Merck Group (1 mentions)
P thr, by Merck Group (1 mentions)
P tyr, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
U0126, by Promega (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Gst antibody, by Cell Signaling Technology (1 mentions)
6 protocols using mg 132
1
Immunocytochemistry Reagents and Protocols
2
Cloning and Transfection of MICB Promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genomic DNA of the A549 cells was isolated and the full-length MICB promoter was amplified using polymerase chain reaction (PCR). The promoter fragments were cloned into the pGL3 luciferase reporter plasmid. The A549 cells were transfected with 0.79 µg MICB promoter plasmid and 0.01 µg SV40-Renilla plasmid using Lipofectamine® 2000 and Plus reagents (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells (5×106) were cultured at 37°C for 24 h, following which they were treated with 10 µM MG132 at 37°C for 8 h and then lysed (Promega Corporation, Madison, WI, USA). Luciferase and Renilla activity were measured as previously described (22 (link)).
3
Detailed Sodium Butyrate Experimental Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sodium butyrate was purchased from Sigma. Pharmacological inhibitors were from Sigma [Actinomycin-D, Cycloheximide, Mithramycin A, H-7, Staurosporin, Rp-CAMP, Sodium orthovanadate (vanadate), Bisindolylmaleimide-I, Go6976, Rottlerin and Okadaic acid], Calbiochem (H-89, U0126, SB203580, JNK-II and MG-132) and Promega (LY294002). Antibodies were purchased from Abcam (TLR5 and Sp1), Santacruz Biotechnology [Sp1 (sc-14027), Sp3 (sc-644 and sc-13018), PKC-δ (sc-213), p300 (sc-584X), p-Ser(4A3) (sc-81516), egr-1 (sc-189), c-myc (sc-789), TLR5 (sc-10742) and tubulin (sc-5286)], Cell Signaling (H3, H4, p-PKC-δ, ERK, p-ERK, p-Thr, p-Tyr, Acetyl-Lys), Sigma (HDAC1) and Upstate (Acetyl-H3 and Acetyl-H4). All others chemicals were purchased from Sigma. Luciferase reporter constructs were bought from Promega. egr-1 overexpression construct and TLR5 promoter deletion reporter constructs were generated in laboratory. pN3-Sp3FL (Addgene plasmid # 24541) and pN3-Sp1FL (Addgene plasmid # 24543) were gifts from Guntram Suske. Primers used for the study are listed in Supplementary Table S1.
4
GST-N-Myc Ubiquitination Assay with p300
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant GST-N-Myc protein was incubated with or without recombinant p300 protein in a ubiquitination reaction containing 20 mM Hepes (pH 7.5, catalog no.: H3375; Sigma–Aldrich), 5 mM MgCl2 (catalog no.: AM9530G; Thermo Fisher Scientific), 2 mM dithiothreitol (catalog no.: R0861; Thermo Fisher Scientific), 2 mM ATP (catalog no.: A6559; Sigma–Aldrich), 5 mg of ubiquitin (catalog no.: U5507; Sigma–Aldrich), 20 mM MG132, and 5 μl/reaction crude rabbit reticulocyte lysate (catalog no.: L4151; Promega) for 1 h at 30 °C (24 (link)). Products were then subjected to IP using GST antibody (catalog no.: 2624; Cell Signaling Technology) and protein A/G magnetic beads before Western blot.
5
Immunocytochemistry with VE-cadherin antibody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse anti-VE-cadherin antibody (sc-6458) diluted 1:50 was obtained from Santa Cruz Biotechnology (United States). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (United States) and Molecular Probes (United States), respectively, and used for immunocytochemistry. Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, United States) and Santa Cruz Biotechnology (sc-3067, United States). Marimastat from Santa Cruz Biotechnology (sc-202223, United States), MG-132 from Promega (G9951, United States). Cytotoxgreen was obtained from Essen BioScience (United States) and CellROXgreen from Molecular Probes (United States). PX-ethyl was purchased from Sigma (United States), according to Sigma safety data sheet safety measures of eyeshields, face shields, full-face respirator and Gloves should be taken. For assessing the BBB response, PX freshly made in ethanol to 400 mM stock solution was immediately diluted in the medium to the desired final concentrations and added to the cell culture. All other reagents applied in this study were used in accordance to the known literature and the supplier's guidelines.
6
Cell-based Assay for Ubiquitination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies to Flag and HA were purchased from Sigma (St. Louis, MO). Anti-Myc, anti-ubiquitin, anti-β tubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GST antibodies, peroxidase-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin were acquired from CWBIO (CWBIO, China). p53 and RNF125 was obtained from Santa cruz (Santa Cruz, CA).
Lipofectamine 2000 transfection reagent, RNase A, RPMI 1640 medium, DMEM culture medium, and fetal bovine serum were from Invitrogen. MG132 was purchased from Promega. Etoposide (Etop) and adriamycin (ADR) were obtained from Sigma. Fetal bovine serum was purchased from Hyclone. Protease inhibitor cocktail was acquired from Roche. The dual luciferase reporter assay kit was obtained from Promega. The GenBank accession number for mRNF125 is AB259692.
Lipofectamine 2000 transfection reagent, RNase A, RPMI 1640 medium, DMEM culture medium, and fetal bovine serum were from Invitrogen. MG132 was purchased from Promega. Etoposide (Etop) and adriamycin (ADR) were obtained from Sigma. Fetal bovine serum was purchased from Hyclone. Protease inhibitor cocktail was acquired from Roche. The dual luciferase reporter assay kit was obtained from Promega. The GenBank accession number for mRNF125 is AB259692.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!