Journal: Scientific Reports

Article Title: Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation

doi: 10.1038/s41598-020-68505-1

Figure Lengend Snippet: Cytotoxicity of pooled human serum against RBCs. There was no cytotoxicity of pooled human serum against TKO pRBCs or human blood type O RBCs. When the concentration of human serum was 25% or less, the cytotoxicity against GTKO pRBCs was significantly lower than that against WT pRBCs (p < 0.01). See “ ” section (Antibody-dependent complement-mediated hemolytic assay). Briefly, RBCs (800 × 10 6 /ml in 75 µl) were isolated and placed in 5 ml round-bottom tubes. Titrated non-heat-inactivated serum (i.e. with complement activity; 450 µl) with PBS and 5% sorbitol (375 µl) instead of IVIg was added to each tube (total 900 µl) and incubated at 37 °C for 150min . Control samples consisted of PBS (instead of the RBC solution) + 450 µl of titrated non-heat-inactivated serum with PBS + 5% sorbitol. After centrifugation at 910 g for 5 min, the supernatant was collected, and each 300 µl was transferred into 96-well plates. The released hemoglobin was measured at an optical density (OD) of 541 nm using a SpectraMax M2e plate reader (Molecular Devices Corp). Data were obtained in duplicate. Results are expressed as mean + /− SD. The dotted line represents cut-off value (7%). (**p < 0.01).

Article Snippet: The released hemoglobin was measured at an optical density (OD) of 541 nm using a SpectraMax M2e plate reader (Molecular Devices Corp. Sunnyvale, CA) .

Techniques: Concentration Assay, Hemolytic Assay, Isolation, Activity Assay, Incubation, Control, Centrifugation

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Cascade assay for target engagement confirms the mechanisms of action for reference compounds. (A) Schematic of MK2/3 signaling. (B) Isolated reactions tested in the biochemical cascade assays. (C) Concentration-response profiles for CC-99677 and ATI-450 against active kinases on their own (open symbols) or for cascade assays using active p38α to activate unactive MK2 or MK3 (solid symbols). Phosphorylation of the respective substrates for the individual enzymes, or in the cascade assays for the unactive enzyme (MK2 or MK3), were measured by HTRF.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Drug discovery, Isolation, Concentration Assay, Phospho-proteomics

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Dual HTRF assay development. (A) Schematic of the dual HTRF assay targeting free MK2 bound to a covalent biotinylated tracer (biotin|cov; see panel (C)). (B) Determination of specific antibody pairs for detecting total MK2. Competition (left) of unlabeled monoclonal antibodies for the epitope of anti-MK2 D1E11 terbium (Tb)-cryptate, enabling the identification of a minimally competitive antibody for quantification of total MK2. Unlabeled antibodies were titrated into reactions containing 0.5× anti-MK2 D1E11 Tb-cryptate and 10 nM biotin|cov tracer paired with streptavidin Alexa Fluor 488. Mouse anti-MK2 7H4.2 was chosen for further assay development and directly conjugated to Alexa Fluor 633 for enhanced HTRF signal. Specificity of this antibody pair for MK2 was tested against recombinant human MK2 or MK3 (right). The antibody pair anti-MK3 D54E4 Tb-cryptate with anti-MK3 2B5 Alexa Fluor 568 was used as a positive control for detection of MK3. Note the cross-reactivity of anti-D1E11 Tb-cryptate for MK3. (C) Structure of biotin|cov, the tracer used for the dual HTRF assays (left). The specificity of this tracer for recombinant human MK2 over MK3 is shown in the associated plot (right), using 0.5× anti-MK2 D1E11-Tb cryptate as the energy donor. (D) CC-99677 target engagement on 10 nM recombinant human MK2 using the dual HTRF assay reagents (anti-MK2 pair plus biotin|cov with streptavidin Alexa Fluor 488). The cause of the observed incomplete target occupancy by this compound is unknown. (E) Quantification of endogenous MK2 protein abundance in human cell lines (left) and murine RAW264.7 cells or splenocytes (right). Specificity of the chosen anti-MK2 pair was demonstrated by the absence of signal from genetic knockout samples (human U937 and mouse splenocytes). HCC1428 possessed the highest detectable concentration of MK2 among the adherent human cell lines (family of red symbols), so it was used for studies to measure endogenous target engagement.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: HTRF Assay, Bioprocessing, Assay Development, Recombinant, Positive Control, Drug discovery, Quantitative Proteomics, Knock-Out, Concentration Assay

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: NanoBRET assay development. (A) Emission spectrum of NanoLuciferase measured live in dimethyl sulfoxide-treated HeLa cells over-expressing either amino- or carboxy-terminal MK2 fusions, with or without 0.17 mM (0.004%) Triton X-100 permeabilization (dashed or solid lines, respectively). The filter band passes used to measure NanoBRET signal are highlighted (centered at 452 nm for NanoLuciferase and 600 nm for BODIPY585). Magnified portion of the spectrum (right) shows the fraction of light from the NanoLuciferase donor that overlaps with –and should be subtracted from– the BODIPY acceptor emission signal. (B) Characterization of BODIPY|rev and BODIPY585|cov tracers, which compete for the same binding pocket as CC-99677. Structures of tracers (left) are shown alongside magnified emission spectra when added to HeLa cells transfected with plasmids encoding either amino- or carboxy-terminal MK2 fusions (middle) as well as the ratiometric NanoBRET signal following a titration of each tracer (right). Signal was measured 1 hour after tracer addition to the live intact (solid) or permeabilized (open/dashed line) cells using a CLARIOstar Plus (average of 5 replicates with standard deviation is shown). Line weight and style follow the legend in panel (A). (C) and (D) NanoBRET signal kinetics in the presence of BODIPY585|cov tracer, using well-by-well acquisitions (C) on the CLARIOstar Plus (CLARIOstar) or full-plate imaging (D) with a GNF Systems luminescence plate reader (LPR). NanoLuciferase signal (blue profiles in shaded background) decayed over time whereas the NanoBRET ratio (red profiles in white background) increased with exposure time to tracer. Continuous measurements for longer than 1 hour were achieved by supplementing with Endurazine substrate. (E) and (F) Measurement of k -on (E) and k -off (F) for CC-99677 using NanoBRET with BODIPY585|cov on a luminescence plate reader. The time points denote when CC-99677-containing medium was exchanged for medium containing excess BODIPY585|cov, which irreversibly quenched the system by saturating free binding sites. The profile for a reversible compound that occupies the same pocket as the covalent CC-99677 (gray, open circles) is included in the k -off plots (F).

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Assay Development, Expressing, Binding Assay, Transfection, Titration, Standard Deviation, Imaging, Microplate Reader Luminescence Measurement

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Comparison of compound target occupancy assays. (A) to (C) Measurement of MK2 target occupancy using dual HTRF, for endogenous protein targeting, in human HCC1428 cells (A) or murine RAW264.7 cells (B) versus NanoBRET in HeLa cells transfected with NanoLuciferase amino-terminally fused to human MK2 (C). Cells were treated with compound for 3 hours before washout. Curve fits are shown for the “free” signal (the ratio of “tracer”/“total”) for CC-99677 treatment, with regression ( R 2 ) values of 0.96 (RAW264.7 in HTRF); 0.92 (HCC1428 in HTRF), and 0.99 (HeLa in NanoBRET). (D) and (E) NanoBRET target occupancy data for 3-hour CC-99677 exposure in the presence of increasing concentrations of fetal bovine or human serum (D) to model in vivo compound availability using the NanoBRET assay. The right-shifted phenotype of CC-99677 is independent of human serum lot (E), suggesting that the free-fraction of CC-99677 compound will be reduced in circulation.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Comparison, Transfection, In Vivo