Z leucyl leucyl leucinal mg 132

Proteasome ChTL activity and CL activity were determined by hydrolysis of fluorogenic substrates Suc-LLVY-AMC (Sigma, USA) and Z-LLE-AMC (Tebu-Bio, Belgium), respectively. The activity was determined in portions of 0.5–2 μl of brain extracts in final volume of 100 μl of reaction mix containing 50 mM Na-HEPES, (pH 7.5), 1 mM DTT, and 30 μM Suc-LLVY-AMC or Z-LLE-AMC.
In Supplementary Figure 1, the time dynamics of proteasome ChTL and CL activities in brain extracts of Wistar and August rats obtained with the use of DTX 880 Beckman Coulter and Multimode Analysis Software is presented. On the basis of the results showing the linear dependence of proteasome activities on reaction time, interval of 20 min was chosen for experiments.
So, the reactions were carried out at 37°C for 20 min and terminated by the addition of 1% SDS. The digestion product was detected by using a fluorimeter with the excitation wavelength of 380 nm and the emission wavelength of 440 nm. Proteasome-independent activity was determined in the presence of 5 μM of inhibitor of proteasome activities, Z-leucyl-leucyl-leucinal (MG-132) (Sigma, USA) (less than 10% activity in all samples) and subtracted from the values obtained in the absence of MG-132. In final, proteasome activities were normalized to 1 mg of protein, detected by Lowry method [43 (link)].

Combined mouse monoclonal antibodies (mAbs) to subunits α1,2,3,5,6,7, mouse mAbs to subunit Rpt6, to immune subunit LMP2 and to immune subunit LMP7, rabbit polyclonal antibodies (pAbs) to immune subunit LMP2 and to immune subunit LMP7 were purchased from Enzo Life Sciences (UK). Mouse mAbs to β-actin, peroxidase-conjugated goat anti-mouse IgG antibodies and peroxidase-conjugated goat anti-rabbit IgG antibodies were purchased from Santa Cruz (Germany). Mouse mAbs to cytokeratin 18 were purchased from Chemicon (USA). ECL reagents were purchased from GE Healthcare (UK). Proteasome substrate N-succinyl-leu-leu-val-tyr7-amido-4-methyl coumarin (Suc-LLVY-AMC) and proteasome inhibitor Z-leucyl-leucyl-leucinal (MG132) were purchased from Sigma (USA). Peroxidase blocking reagent, LSAB2 System–HRP, mouse mAbs to ERα (clone 1D5), mouse mAbs to PR (clone PgR 636) and biotinilated anti-mouse IgG antibodies were purchased from Dako (Denmark). Hoechst 33342, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 donkey anti-mouse IgG were purchased from Invitrogen (USA).