Caspases,Paracaspases, and Metacaspases Methods and Protocols

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5. Measure the fl uorescence (excitation 350–380 nm, emission
   460 nm) over time (measure every 10 min for 2–4 h) at 30 °C.


6. To get the MALT1 protease activity, calculate the slope of the
   fl uorescence over time and normalize to the amount of MALT1
   protein ( see Note 11 ).

The detection of MALT1 substrate cleavage or MALT1 monou-

biquitination can serve as a qualitative means to monitor MALT1

activity. The MALT1 mediated cleavage of A20, CYLD, RelB, and

Regnase can easily be visualized by standard western blot [ 17 , 20 ,

24 , 25 ]. Only the detection of cleaved human BCL10 is challeng-

ing since MALT1 removes only fi ve amino acids from its

C-terminus. Cleaved BCL10 can therefore only be detected using

high-resolution gels [ 16 ] or an antibody specifi c for cleaved BCL10

[ 32 ]. Moreover, the 8 kDa shift caused by the attachment of a

single ubiquitin to MALT1 is more easily detectable using high-

resolution gels. Here, we describe only the preparation of the

high-resolution gels [ 34 ] for detection of cleaved BCL10 and

MALT1 monoubiquitination since the further procedure does not

vary from standard SDS-PAGE and western blot protocol.

1. To prepare a 500 ml stock solution of a high resolution 15 %
   acrylamide resolving gel mix to detect cleaved BCL10, add
   250 ml acrylamide (30 % w/v), 43 ml bis-acrylamide (1 % w/v),
   125 ml resolving buffer, and 82 ml water. For a 7,5 % high-
   resolution acrylamide gel mix, which is optimal to detect
   MALT1 monoubiquitination, add 125 ml acrylamide
   (30 % w/v), 97 ml bis-acrylamide (1 % w/v), 125 ml resolving
   buffer, and 150 ml water ( see Note 12 ).


2. Add 50 μl of 10 % ammonium persulfate and 5 μl of TEMED
   per 10 ml of mix. Cast the gels within minutes and gently over-
   lay with isopropanol.


3. Proceed with standard SDS-PAGE and western blot protocol.

To quantify MALT1 protease activity in intact cells we generated an

eYFP–Leu-Val-Ser-Arg–eCFP expression construct as well as the

respective non-cleavable negative control, eYFP–Leu-Val-Ser- Gly–

eCFP [ 31 ]. Here we describe the use of the FRET-based cleavage

assay in HEK293T cells, but it might be adapted to other cells.

1. Plate approximately 150.000 HEK293T cells in DMEM (with
   10 % FCS and antibiotics) in a 6-well cell culture plate.


2. After 24 h, transfect the sub-confl uent HEK293T cells with
   0.1 μg of the reporter construct together with 1 μg MALT1
   and 0.2 μg BCL-10 expression constructs using the calcium
   phosphate transfection method. Mix 125 μl water with 12.5 μl
   CaCl 2 (2.5 M), add the respective amount of plasmids and add
   125 μl 2× HeBS dropwise while gently shaking on a vortex.

3.3 Detection of
Endogenous BCL10
Cleavage or MALT1
Monoubiquitination by
High-Resolution Gels
and Western Blot

3.4 FRET-Based
Assay of Protease
Activity

Stephan Hailfi nger et al.

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