Prolyl endopeptidase (IC50 = 6.3 μM)； Sulfhydryl (-SH) groups of proteins [2]

N-Ethylmaleimide (20 μM;30 min) prevents the phosphorylation of Akt Ser-473, Akt Thr-308, p70S6K, ribosomal protein S6, 4E-BP1, eIF4E, BAD, and FKHR-L1[2].
N-Ethylmaleimide (20 μM;30 min) affects conversion of pro-caspase-3 in vascular smooth muscle cells[2].
N-Ethylmaleimide (20 μM;6 h) induces apoptosis in vascular smooth muscle cells[2].
N-Ethylmaleimide (20 μM;30 min) has an impact on ROS production and PP2A activity in vascular smooth muscle cells[2].

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N-ethylmaleimide (NEM) was used to block sulfhydryl groups in cell experiments. Treatment with NEM (100 μM) inhibited the neddylation process, as indicated by the accumulation of unneddylated Cullin1 in HEK293T cells. This inhibition affected the β-catenin degradation pathway in the Wnt signaling, leading to the accumulation of β-catenin and increased expression of its target genes such as AXIN2 and MYC [1]

NEM (10 mg/kg) was administered intraperitoneally to rats, and it reversed the gastroprotective effect of momordin Ic on ethanol-induced gastric mucosal lesions. This reversal was associated with the depletion of sulfhydryl groups, as NEM reacts with sulfhydryl groups to form covalent adducts, thereby inhibiting the protective role of endogenous sulfhydryls in gastric mucosa [2]
N-Ethylmaleimide (10 mg/kg; i.h.) increases the prevalence situation in mice with acute gastric ulcers[3].

HEK293T cells were seeded and cultured, then treated with NEM (100 μM) for a certain period. Cell lysates were prepared, and Western blot analysis was performed to detect the levels of neddylated and unneddylated Cullin1, as well as β-catenin and its target gene products (AXIN2, MYC). The results showed that NEM inhibited neddylation and affected β-catenin degradation [1]

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Cell lysates are prepared in Pierce IP Lysis Buffer, which contains protease inhibitors, phosphatase inhibitors, and the deubiquitinating inhibitor N-Ethylmaleimide (NEM, 10mM), in order to facilitate endogenous protein immunoprecipitation. The lysate supernatant is first incubated for 14 hours at 4 °C with a primary antibody, and then it is combined with Dynabeads Protein G magnetic beads and left to rotate for 4 hours at 4 °C. After using a magnetic separation rack to separate the magnetic bead-Ab complex, PBST buffer is used three times to wash the beads. The complex-bound beads are resuspended in 1x protein electrophoresis loading buffer and heated to 100 °C for 8 minutes, after which the supernatant is collected for SDS-PAGE analysis. Before preparing cell lysates for exogenous protein immunoprecipitation, cells are transfected for 48 hours. Antibody against mouse IgG is used in parallel to prepare the negative control.

Rats were fasted for 24 hours before the experiment. NEM was dissolved in a suitable solvent and administered intraperitoneally at a dose of 10 mg/kg. After 30 minutes, momordin Ic was given orally, followed by ethanol administration 30 minutes later to induce gastric mucosal lesions. The rats were sacrificed after a certain period, and the gastric mucosa was examined for lesion formation [2]

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Male Sprague-Dawley rats
10 mg/kg
s.c.

Adverse Reactions:
Skin Toxins - Skin burns.
Lacrimal Agents - Substances that irritate the eyes and induce tear production.
Toxic Pneumonia - Inflammation of the lungs caused by inhalation of metallic fumes or toxic gases and vapors.

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4362 rat oral LD50 25 mg/kg Behavior: somnolence (overall activity inhibition); Behavioral: seizures or effect on epileptic threshold National Technical Information Service, OTS0536969
4362 rat LD50 intraperitoneal injection 1 mg/kg Behavior: somnolence (overall activity inhibition) National Technical Information Service, OTS0536969
4362 rat LDLo intracerebral injection 1669 μg/kg Journal of Medicinal Chemistry, 15(534), 1972
4362 mouse LD50 oral 25 mg/kg Behavior: somnolence (overall activity inhibition) National Technical Information Service, OTS0536969
4362 mouse LD50 intraperitoneal injection 5 mg/kg Behavior: somnolence (overall activity depression) National Technical Information Service, OTS0536969

[1]. Neddylation is essential for β-catenin degradation in Wnt signaling pathway. Cell Rep. 2022 Mar 22;38(12):110538.

[2]. Roles of capsaicin-sensitive sensory nerves, endogenous nitric oxide, sulfhydryls, and prostaglandins in gastroprotection by momordin Ic, an oleanolic acid oligoglycoside, on ethanol-induced gastric mucosal lesions in rats. Life Sci. 1999;65(2):PL27-32.

N-Ethylmaleimide is a maleimide compound, specifically an N-ethyl derivative of maleimide. It functions as an inhibitor of EC 2.1.1.122 [(S)-tetrahydroproberonine N-methyltransferase], EC 2.7.1.1 (hexokinase), EC 1.3.1.8 [acyl-CoA dehydrogenase (NADP(+))], and anti-coronavirus drugs. Its function is similar to that of maleimide. It is a widely used thiol reagent in experimental biochemical research. β-catenin is a core component of the Wnt signaling pathway; its degradation is closely related to ubiquitination, but it is rarely studied through loss-of-function experiments. We observed that removing endogenous β-catenin ubiquitination using the ubiquitination inhibitor TAK-243 did not improve the stability of β-catenin. We demonstrated that the specific NEDDylation inhibitor MLN4924 rapidly and significantly stabilized N-terminal phosphorylated β-catenin across all cell types examined, and that inhibition of NEDDylation, rather than ubiquitination, significantly enhanced the interaction between β-catenin and TCF4. We also confirmed that the E3 ligase β-TrCP2 (but not β-TrCP1) is associated with NEDDylation and degradation of β-catenin. GSK3β and adenomatous polyposis protein (APC) are not essential for β-catenin NEDDylation, but are crucial for its subsequent degradation. Our findings not only elucidate the modification and degradation of β-catenin in the Wnt signaling pathway but also underscore the importance of re-evaluating previously identified ubiquitination substrates. [1]

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This study investigated the effects of oleanolic acid oligosaccharide—matrine Ic—isolated from the fruit of Kochia scoparia (L.) SCHRAD., on capsaicin-sensitive sensory nerves (CPSN), endogenous nitric oxide (NO), sulfhydryl groups (SH), and prostaglandins (PG) in ethanol-induced gastric mucosal injury in rats. The results showed that matrine Ic (10 mg/kg, orally) could inhibit ethanol-induced gastric mucosal injury. The effect of matrine Ic was significantly weakened after pretreatment with capsaicin (total dose 125 mg/kg, subcutaneously, a CPSN inhibitor), N(G)-nitro-L-arginine methyl ester (L-NAME, 70 mg/kg, intraperitoneally, a NO synthase inhibitor), N-ethylmaleimide (NEM, 10 mg/kg, subcutaneously, an SH receptor blocker), or indomethacin (10 mg/kg, subcutaneously, a PG biosynthesis inhibitor). L-arginine (300 mg/kg, intravenously, a NO synthase substrate) eliminated the attenuating effect of L-NAME, but D-arginine (300 mg/kg, intravenously, an enantiomer of L-arginine) did not. The combined use of capsaicin with indomethacin, NEM or L-NAME was not more effective than capsaicin alone. The combined use of indomethacin with NEM, indomethacin with L-NAME, or indomethacin, NEM and L-NAME enhanced the inhibitory effect when used alone. These results suggest that CPSN plays an important role in the protective effect of momordicin Ic against ethanol-induced gastric mucosal damage, and that endogenous prostaglandins (PGs), nitric oxide (NO) and thiol groups (SHs) interact in mice. [2] NEM is a thiol-reactive compound that can covalently modify free thiol groups in proteins, thereby inhibiting their function. In the Wnt signaling pathway, NEM interferes with NEDDylation modification by targeting thiol groups in related proteins, thereby interfering with the degradation of β-catenin[1]. In studies on gastric mucosal protection, NEM has been used as a tool to deplete endogenous thiol groups, which helps to study the role of thiol groups in the gastric mucosal protection mechanism[2].

C6H7NO2

125.13

125.047

C, 57.59; H, 5.64; N, 11.19; O, 25.57

128-53-0

N-Ethylmaleimide-d5;360768-37-2

4362

White to light yellow solid powder

1.2±0.1 g/cm3

210.0±9.0 °C at 760 mmHg

43-46 °C(lit.)

73.3±0.0 °C

0.2±0.4 mmHg at 25°C

1.514

0.68

0

2

1

9

166

0

O=C1C([H])=C([H])C(N1C([H])([H])C([H])([H])[H])=O

HDFGOPSGAURCEO-UHFFFAOYSA-N

InChI=1S/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3

1-ethylpyrrole-2,5-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (16.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (16.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (16.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (799.17 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)