Streptavidin coated plate

The ELISA for assessing the response to Fusion-Peptide (FP8) was conducted with 96-well streptavidin-coated plates (Thermo Fisher) and biotinylated eight-residue fusion peptide (FP8-PEG-biotin). Subsequently, the plates were blocked with B3T buffer (comprising of 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 3.3% fetal bovine serum, 2% bovine albumin, 0.07% Tween 20, and 0.02% thimerosal) prepared in-house. The serum was serially diluted at 7-point 5-fold dilution and incubated for 1hr. Goat anti-NHP HRP conjugated secondary was added and incubated for an hour. The plates were then developed for 10 minutes using tetramethylbenzidine (TMB) substrate (SureBlue; KPL, Gaithersburg, MD). To stop the reaction, 1 N H₂SO₄ sulfuric acid was added. Finally, the plates were read on a microplate spectrophotometer (Biotek Epoch, Winooski, VT) to determine the endpoint titer for each sample.⁸

Cells expressing K_V10.1-BBS (52 (link)) were incubated for 10 min on ice with 2.5 μg/ml α-bungarotoxin (BTX)-biotin (Invitrogen), washed twice with ice-cold PBS, scraped, and centrifuged at 800 × g for 3 min. Cell pellets were resuspended in 20 mm Tris-HCl, 150 mm NaCl, 5 mm MgCl₂, and 1% Nonidet P-40, pH 7.4, containing protease inhibitors. The lysates were passed several times through a 25-gauge needle, placed in microcentrifuge tubes, incubated for 20 min on ice, and finally centrifuged at 16,100 × g for 15 min at 4 °C. Streptavidin-coated plates (Thermo Scientific) were washed twice with PBS containing 0.05% Tween 20, 0.1% BSA, and 0.1% Triton X-100. 30 and 150 μg of protein (in triplicates) were then incubated for 30 min on ice. The plates were washed twice and then blocked for 30 min with NPE (150 mm NaCl, 5 mm EDTA, 50 mm Tris, 5 mm KCl, and 1% Nonidet P-40, pH 7.5) containing 1% casein. Anti-K_V10.1 antibody (mAb62) was diluted in NPE with 0.1% casein and used at 2.5 μg/ml for 90 min. The wells were washed three times with NPE with 0.1% casein and blocked again in NPE with 1% casein for 30 min. Secondary antibody (1:500 ECL HRP-linked anti-mouse IgG (GE Healthcare)) was then incubated for 90 min. After seven washes with NPE-0.1% casein, peroxidase activity was determined using ABTS as described above.

Animal sera and monoclonal antibodies were assessed for binding to a biotinylated eight amino acid linear peptide FP8-PEG12-biotin (GenScript, Piscataway, NJ). Streptavidin coated plates (Thermoscientific, Rockford, IL) were washed with PBS-T buffer (10% Tween-20 in 1X PBS) then incubated with FP8v1-PEG12-biotin at 37 °C for 2 hours. Animal sera were heat-inactivated at 56 °C for 1 hour and assessed at 7-point 4-fold dilutions starting at 1:25 dilutions. Monoclonal antibodies were tested at 7-point 5-fold dilutions starting at 5 μg/ml. Samples were first incubated on plates at 37 °C for 1 hour, then the plates were washed with PBS-T buffer. Secondary antibody (goat anti-guinea pig IgG HRP) was added and incubated in wells at 37 °C for 1 hour before the final PBS-T buffer wash. TMB substrate (SureBlue, KPL, Gaithersburg, MD) was equilibrated to ambient temperature and incubated in wells for 10 minutes, when the reaction was stopped with 1 N sulfuric acid. The plates were then read on a microplate spectrophotometer (Biotek Epoch, Winooski, VT) and the endpoint titer for each sample was determined.

B-cell linear library ELISA was performed in a similar manner to a previously established peptide-based screen¹⁸ . In brief, streptavidin‐coated plates (Thermo Fisher Scientific, #15125) were blocked with 0.1% PBST (0.1% v/v Tween‐20, Sigma-Aldrich, #P1379-500ML, in PBS, Gibco, #20012-043) containing 1% w/v sodium caseinate (Sigma‐Aldrich, #C8654-500G, lot BCBP6469) and 1% w/v bovine serum albumin (BSA; Sigma‐Aldrich, #A7030-500G, lot SLBW5033) overnight at 4 °C, before addition of pooled or single biotinylated peptides at 1:1000 dilution in 0.1% PBST. Heat‐inactivated patient serum samples were added at 1:1000 dilution in 0.1% PBST. Horseradish peroxidase-conjugated goat anti-human IgG (H + L) antibody (Jackson ImmunoResearch, #109-035-088, lot 139159) prepared in 10% blocking buffer was used for detection of peptide‐bound antibodies. In total, 100 μL of TMB substrate (Sigma‐Aldrich, #T8665, lot SLCB5343) was used for a 5 minute development and was stopped by addition of 100 μL of 0.16 M sulfuric acid prepared from 95% to 97% Sulfuric Acid stock solution (Merck, #1.00731.1000), prior to absorbance measurements. Absorbance was measured with the following parameters: 450 nm minus 690 nm (bandwidth of 9 nm) in five flashes after a 10 second shaking at 1 mm amplitude on an Infinite M200 plate reader (Tecan, firmware V_2.02_11/06).

Activity of MALT1 was determined as described earlier [22 ]. To this end DLBCL cell lines (5 × 10⁵/ml per sample) were treated with different concentrations of Ibrutinib and S-Mepazine for 18 h and lysed in 130 μl co-IP buffer (25 mM hepes pH 7.5, 150 mM NaCl, 10% glycerol, 0.2% NP-40, 1mM DTT, 10 mM sodium fluoride, 8 mM β-glycerophosphate and 300 μM sodium vanadate) w/o protease inhibitors. After centrifugation and removal of 10 μl input control, extract was incubated with 0.1 μM biotinylated ABP (activity-based probe) at RT for 1 h to form active MALT1-ABP complexes. Extract was transferred to streptavidin-coated plates (Thermo Scientific) and incubated o/n at 4°C. Plates were washed with PBS-T (0.05% Tween), blocked with 2% BSA and incubated with primary MALT1 antibody (2494; Cell Signaling) for 1h at RT. After further washing, HRP-conjugated secondary antibodies were added for 1 h, washed and TMB substrate was incubated for approximately 15 min. Reaction was stopped with 1M H₂PO₄ and luminescence was analyzed in a Biotek reader at 450 nm (background 570 nm).