Surepage bis tris

To visualize the protein profiles of total cell extracts or fractions of it, proteins were separated according to their size in a 4–20% acrylamide gel (SurePAGE Bis-Tris, Genscript #M00655) and then silver-stained. The gel was fixed with a fixative solution (40% (v/v) ethanol, 10% (v/v) acetic acid) for 30 min and incubated overnight with a solution containing 30% (v/v) of ethanol, 0.26% (v/v) of glutaraldehyde, 6.8% (w/v) of sodium acetate trihydrate and 0.2% (w/v) of sodium thiosulfate pentahydrate. After three washes of 5 min in deionized water, the gel was incubated for 40 min with a solution containing 0.1% (w/v) of silver nitrate and 0.1% (v/v) of formaldehyde. After a brief wash of 10 s in deionized water, the gel was incubated 1–5 min with a developmental solution containing 2.5% (w/v) of sodium carbonate and 0.01% (v/v) of formaldehyde until the appearance of proteins. The developmental reaction was stopped by the incubation of the gel with a solution containing 1.46% (w/v) of EDTA dihydrate.

10⁶D. discoideum cells were pelleted and resuspended in 40 µl of reducing sample buffer [20.6% (w/v) sucrose, 100 mM Tris pH6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS, 6% (v/v) β-mercaptoethanol]. Twenty µl of each sample was migrated (200 V, 30 min) in a 4–20% acrylamide gel (SurePAGE Bis-Tris, Genscript #M00655), and transferred to a nitrocellulose membrane using a dry transfer system for 7 min (iBlot gel transfer device, Invitrogen #IB1001EU). The membranes were blocked during 2 h in PBS containing 0.1% (v/v) Tween 20 and 7% (w/v) milk, and washed three times for 5 min in PBS + 0.1% (v/v) Tween 20. The AL626 antibody specific for the ALFA epitope was produced as a mini-antibody (VHH-mouseFc) by the Geneva Antibody Facility (https://www.unige.ch/medecine/antibodies/). The membranes were incubated with the primary antibody AL626 (dilution 1:50) (Guilhen, 2020 (link); Lima and Cosson, 2020 (link)) for 1 h at room temperature, then washed three times for 5 min. The membranes were then incubated with horseradish peroxidase-coupled goat anti-mouse IgG (Biorad #170-6516, dilution 1:3,000) or anti-rabbit IgG (Sigma-Aldrich #A8275, dilution 1:3,000 and washed twice for 5 min and once for 15 min in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Millipore) using a PXi-4 gel imaging systems (Syngene).

Sequences from the unique variable regions were synthesized with adaptors for cloning at GenScript (China). Cloning was performed using in-house vectors for expression. Plasmids were transfected into human embryonic kidney 293T cells (ATCC) using PEI (Sigma, #24885) in a 24-well plate and incubated at 37°C, 5% CO₂ for 3 days before collecting supernatant for subsequent test. Positive clones in supernatant test were transfected into HEK 293F (Gibco, USA, R79007) or ExpiCHO cells (Thermo Fisher Scientific, A29127) for small-scale recombinant antibody production. Antibody was purified using Protein A (AmMag Protein A Magnetic Beads, Genscript, L00695) affinity chromotography, and purity was measured by both SDS-PAGE (SurePAGE, Bis-Tris, 10 × 8, 4% to 12%, 12 wells, Genscript, M00653) and SEC-HPLC (GE Healthcare).

Frozen cell pellets were lysed in a buffer of 50 mM Tris–HCl, pH 7.5, 25% sucrose, 2 mM EDTA, 1 mM DTT, 1 mM ATP, 0.05% digitonin. Proteasome activity was measured in extracts of treated cells with Suc-LLVY-amc (ß5c and LMP7), Ac-ANW-amc (LMP7), Ac-WLA-amc (ß5c), Ac-nLPnLD-amc (ß1c and LMP2), Ac-APL-amc (LMP2) and Ac-RLR-amc (ß2c and MECL1) fluorogenic substrates^{36 (link)}. It was normalized to the total protein content of the extract, which was determined using the Coomassie Plus—The Better Bradford Assay Reagent (Thermo). To distinguish between contribution of ß5c and LMP7 to the cleavage of Suc-LLVY-amc, extracts were preincubated with a highly LMP7-specific inhibitor LU-015i (5 μM) for 30 min at 37 °C immediately before activity measurements. Alternatively, occupancy of active sites was measured with activity-based probes as described^{10 (link),37 (link)}. In a three-probe-cocktail, ß1c (PSMB6) and LMP2 (ß1i) subunits are labeled with Cy5-NC-001, BODIPY(FL)-LU-112 labels MECL1 (ß2i) and ß2c (PSMB7), and LMP7 (ß5i) and ß5c (PSMB5) subunits were labeled with BODIPY(TMR)-NC-005-vs^{10 (link)}. In a two-probe combination, LMP7, ß5c, MECL1 and ß2c subunits were labeled with BODIPY(TMR)-NC-005, and ß1c and LMP2 were labeled with BODIPY(FL)-NC-001^{37 (link),47 (link)}. Labeled subunits were resolved in 10% Bis–Tris SurePAGE™ (Genscript) and imaged on c600 gel imager (Azure Biosystems).

Nuclear extracts were prepared using NE-PER™ nuclear extraction reagent (Thermo Fisher) separated on 4–12% Bis–Tris SurePAGE™ (Genscript) using MOPS running buffer, and transferred to a nitrocellulose membrane using methanol-free transfer buffer^{48 (link)}. The MLL–AF4 fusion protein was revealed using D2M7U antibody to N-terminal antigen of MLL-1; histone H3 was detected with D2B12 antibody (Cell Signaling). We used HRP-conjugated secondary antibodies (Cell Signaling) and Super Signal West Femto Maximum Sensitivity substrate (Thermo Scientific) for band visualization.