Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, Expressing

Journal: F1000Research

Article Title: An unexpected effect of TNF-α on F508del-CFTR maturation and function

doi: 10.12688/f1000research.6683.2

Figure Lengend Snippet: Differentiated primary HBE cell cultures grown at an air-liquid interface were incubated with 50ng/ml TNFα for 10–30min, 3–6h and 24h. CFTR immunodetection was performed with 24.1 anti-CFTR antibody and analyzed with confocal microscopy. Green staining represents CFTR (Alexa Fluor 488), red color staining represents ZO-1 protein of tight junctions (Alexa Fluor 594) and blue DAPI staining visualizes nuclei. Independent TNFα treatments and CFTR immunodetection were performed on HBE cells from three different F508del/F508del CF patients. Representative images of one experiment are demonstrated (Scale bars = 20µm).

Article Snippet: Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031).

Techniques: Incubation, Immunodetection, Confocal Microscopy, Staining

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Figure 6. Histone H2A ubiquitination accompanied by BRCA1 activation is the hallmark phenotype of BRAP LOF (A) Immunoblotting of histone extracts from NPCs as well as from embryonic, neonatal, adult cerebral cortical tissues, and quantification (Mean G SD) of increases in histone H2Aub (total H2Aub and H2AubK119, respectively) resulted from Brap LOF. n = 3–6 biological replicates. p-values calculated by Student’s t test are indicated. (B) Immunoblotting of Brca1 in various cells and tissues, showing that Brap LOF results in increased Brca1 abundance. (C) Immunoblotting of nuclear vs cytoplasmic fractions of MEFs at P1, showing increased nuclear localization of Brca1 in Brap/ cells. (D) Brca1 (red) and NeuN (green) double immunohistology images of cerebral cortical sections of BrapcKONPC and control mice at four months of age. Representative images are shown. Note the increased intensity and density of Brca1 puncta in the nuclei of BrapcKONPC cortical neurons (NeuN+). (E and F) Immunoblotting analyses of histone extracts from cerebral cortical tissues of three-month-old mice, showing increased ubiquitination of H2A variants targeted by Brca1 (E) along with total histone H2A ubiquitination (F). (G) Double immunohistology staining of cortical sections of 4-month old WT or BrapcKONPC mice with antibodies against Gfap (green) and histone H3 (red), showing reduced nuclear histones in cells surrounded by reactive astrocytes (circles) in BrapcKONPC cortical tissues. Representative images are shown. Nuclear DNA was stained with Hoechst 33342. Bars: 50 um or as indicated. See also Figure S3.

Article Snippet: Phospho-p53 (Ser15) Abcam Cat# ab1431; RRID:AB_301090 Phospho-p53 (Ser15) (D4S1H) Cell Signaling Technology Cat# 12571; RRID:AB_2714036 Phospho-p53 (Ser15) (16G8) Cell Signaling Technology Cat# 9286; RRID:AB_331741 Phospho-ATM(Ser1981) Novus Biologicals Cat# AF1655 Phospho-ATR (Ser 428) Cell Signaling Technology Cat# 2853; RRID:AB_2290281 53 BP1 Novus Biologicals Cat# NB100-304 Brca1 Santa Cruz Biotechnology Cat# sc-642; RRID:AB_630944 Brca1 Novus Biologicals Cat# MAB22101 Brca1 (clone 8F7) Novus Biologicals Cat# NBP1-41186 LC3B (G-9) Santa Cruz Biotechnology Cat# sc-376404; RRID:AB_11150489 LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 LC3B Novus Biologicals Cat# NB100-2220 p62SQSTM1 Novus Biologicals Cat# MAB8028 p62SQSTM1 GeneTex Cat# GTX100685; RRID:AB_2038029

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Staining

Journal: Journal of experimental zoology. Part A, Ecological and integrative physiology

Article Title: IS AQUAPORIN-3 INVOLVED IN WATER-PERMEABILITY CHANGES IN THE KILLIFISH DURING HYPOXIA AND NORMOXIC RECOVERY IN FRESHWATER OR SEAWATER?

doi: 10.1002/jez.2393

Figure Lengend Snippet: Killifish were acclimated to either seawater or freshwater, and subjected to normoxia (the control) or 3 h hypoxia (10% O2 saturation), after which the gills were excised. Immunohistochemistry was used to identify the membrane localization of AQP3 (in red), CFTR (in green), and NKA (in blue), with immunofluorescent antibodies, in seawater normoxia (A, B, and C) or hypoxia (D, E, and F) and in freshwater normoxia (G, H, and I) or hypoxia (J, K, and L). All immunofluorescent images of AQP3/CFTR are in 60x and those of NKA are in 40x. AQP3 immunofluorescence generally shared the same distribution as NKA; AQP3 was localized to the basolateral membranes (Ba; example shown on Panel C) of the cells of both the gill lamellae (L), and interlamellar regions (IL) of the filaments (F). AQP3 might also occur on the apical membranes at the outer borders of the lamellae. CFTR immunofluorescence was poor in the freshwater gills, but was clearly discernible in the apical membrane of the interlamellae of seawater gills. NKA immunofluorescence was expressed on the basolateral membranes of the cells of the lamella and interlamellae under all treatments, and was particularly prominent in the interlamellar regions of the seawater gill under normoxia. Ionocytes (inset on Panel C) were apparent in the interlamellar regions of seawater gills under normoxia and hypoxia and were distinguishable by an apical crypt (ApC; Panels C and F) expressing CFTR and the basolateral membrane expressing NKA.

Article Snippet: The primary antibodies were monoclonal mouse CFTR (cat. no. MAB25031, R&D Systems, Minneapolis, MN, USA) and polyclonal rabbit NKA (cat. no. SC-28800, Santa Cruz Biotechnology).

Techniques: Control, Immunohistochemistry, Membrane, Immunofluorescence, Expressing

Journal: Current eye research

Article Title: Immunohistochemical Detection of CTGF in the Human Eye.

doi: 10.3109/02713683.2016.1143014

Figure Lengend Snippet: Figure 2. (A) CTGF-immunoreactivity (A, red) was detected in the corneal epithelium, mainly concentrated in basal layers (blue: DAPI). (B) Single superficial epithelial cells were detected with rather intense immunoreactivity for CTGF (red, arrowhead; blue: DAPI). (C) CTGF-immunoreactivity (red) was also detectable in corneal endothelial cells (arrows), as well as in keratinocytes of the stroma (arrowheads; blue: DAPI). (D) CTGF-immunoreactivity was absent in corresponding negative controls (here: corneal epithelium and stroma; blue: DAPI). (E, F) Conjunctival epithelial cells displayed immunoreactivity for CTGF (E), but CTGF-immunoreactivity was absent in the corresponding negative control (F). Arrowheads: goblet cells; blue: DAPI). (G, H) In the trabecular meshwork (asterisk) and Schlemm’s canal (arrowheads), CTGF immunoreactivity (red) revealed an identical staining pattern (G) as compared with corresponding negative controls (H).

Article Snippet: After a 5 min rinse, slides were incubated overnight at RT with an anti-human connective tissue growth factor (CTGF; raised in goat, 1:100, AF660, R&D Systems, Minneapolis, MN, USA) in TBS, containing 1% BSA and 0.5% Triton X-100.

Techniques: Negative Control, Staining

Journal: Current eye research

Article Title: Immunohistochemical Detection of CTGF in the Human Eye.

doi: 10.3109/02713683.2016.1143014

Figure Lengend Snippet: Figure 3. (A, B) In the iris, CTGF-immunoreactivity (A, red) was present in anterior layers of the iris (arrows) as well as muscle fibers of the iris sphincter (arrowheads), as detected with alpha-smooth-muscle actin (green), but was absent in the corresponding negative control (B). DAPI: white. (C, D) In C, iris vessels displayed immunoreactivity for CTGF (red) in the vascular endothelium (blue, CD31), as seen by an association of both signals (purple color), while an overlap of CTGF with vascular smooth muscle cells (green, ASMA) was not observed, as seen by absence of yellow-mixed color. Immunoreactivity was absent in corresponding negative controls (D). Asterisks indicates vessel lumen; DAPI: white. (E, F) CTGF-immunoreactivity (E, red) was present in the muscle fibers of the ciliary body (asterisk), as detected with alpha – smooth muscle actin (green), but was absent in the corresponding negative control (E). DAPI: white. (G, H) CTGF-immunoreactivity was present in the non-pigmented ciliary epithelium (G, arrowheads), but was absent in corresponding negative controls (F; red signal here, arrows, corresponds to autofluorescence). DAPI: white. (I, J) CTGF-immunoreactivity (red) in the lens was absent in the lens epithelium (I, arrowheads), but present in superficial layers of the lens (I, asterisk), while it was absent in the corresponding negative control (J, asterisk). DAPI: blue.

Article Snippet: After a 5 min rinse, slides were incubated overnight at RT with an anti-human connective tissue growth factor (CTGF; raised in goat, 1:100, AF660, R&D Systems, Minneapolis, MN, USA) in TBS, containing 1% BSA and 0.5% Triton X-100.

Techniques: Negative Control

Journal: Current eye research

Article Title: Immunohistochemical Detection of CTGF in the Human Eye.

doi: 10.3109/02713683.2016.1143014

Figure Lengend Snippet: Figure 4. (A, B) In the retina, CTGF-immunoreactivity (red) was present in the nerve fiber layer (NFL; A: cross-section close to the optic nerve head), and further a weak signal was also present in the IPL and OPL, while immunoreactivity was absent in corresponding negative controls (F). DAPI: blue. (C, D) In the choroid, CTGF- immunoreactivity (red) was present in the choriocapillaris (C, arrowheads) and in blood vessels of the choroidal stroma (C, arrows), but was absent in corresponding negative controls (D). DAPI: blue; RPE: retinal pigment epithelium; CC: choriocapillaris. (E, F) In the optic nerve head, CTGF-immunoreactivity (red) was present in endothelial cells of the central retinal artery (E, asterisks) and was detected in single cells within connective tissue strands of the optic nerve (F, arrowhead; inset represents magnified situation in F). DAPI: blue. All images in Figures 2, 3, and 4 represent confocal images in single optical section mode.

Article Snippet: After a 5 min rinse, slides were incubated overnight at RT with an anti-human connective tissue growth factor (CTGF; raised in goat, 1:100, AF660, R&D Systems, Minneapolis, MN, USA) in TBS, containing 1% BSA and 0.5% Triton X-100.

Techniques:

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–C) MEFs subjected to SPARKL analysis measuring real-time labeling with fluorescently-tagged Annexin V (100 μg/ml) via imaging with an IncuCyte ZOOM. Left panels: kinetics of cell labeling in response to increasing concentrations of 2t–hexadecenal (2t–16) with DMSO vehicle or co-treated with ABT-737 (ABT); black line reports untreated control. Right panels: endpoint data of replicates at 24 hours. Data shown are the mean of technical triplicates and error bars report SEM. (A) WT MEFs (matched to Bax −/− Bak −/− double knockout MEFs) were treated with 2t–16 (10, 20, 40 μM) and DMSO or ABT–737 (1 μM), imaged every 2 hours, and quantified for number of Annexin V-positive objects. (B) Same as in A with Bim −/− Bid −/− double knockout MEFs. (C) Same as in A with Bax −/− Bak −/− double knockout MEFs. (D–I) LUV permeabilization studies with recombinant BAX protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from liposomes. Grey data report LUVs solubilized with 1% CHAPS to measure maximal signal. Data shown are the mean of technical replicates. (D) BAX protein (120 nM) was combined with DMSO vehicle or 2t–16 (6.5–50 μM) followed by addition of LUVs and measured by fluorescent spectroscopy. (E) Same as in D with BIM–BH3 peptide (2.5 μM) added to BAX and 2t–16. (F) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX (120 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figures S1C . (G–H) LUV permeabilization studies as in D–E with BAX (160 nM) and hexadecanal (6.5–50 μM) ± BIM–BH3 peptide (2.5 μM). (I) Heatmap visualization of normalized endpoint LUV permeabilization data as in F with BAX (160 nM) and 16CHO (6.5−50 μM). Data summarized from Figures S1D . See also .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Imaging, Control, Double Knockout, Recombinant, Fluorescence, Liposomes, Spectroscopy, Incubation

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–D) LUV permeabilization studies with recombinant BAX protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from compromised liposomes. Left panels: kinetic fluorescence data; right panels: endpoint data normalized to LUV fluorescence and maximal signal generated by LUVs solubilized with CHAPS detergent (grey data). Data are shown as the mean of technical replicates and error bars report SEM. (A) BAX protein (100 nM) was activated by BIM–BH3 peptide (0.13–2 μM) and added to LUVs. (B) LUVs treated with 2t–16 (16.5–50 μM) in the absence of BAX to confirm no membrane destabilization by 2t–16. (C) Data summarized by . LUVs permeabilized by BAX (120 nM) treated with 2t–16 (6.5– 50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). (D) Data summarized by . LUVs permeabilized by BAX (160 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM).

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Recombinant, Fluorescence, Liposomes, Generated, Membrane

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–B) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated as indicated, and subjected to MST. Data shown are the mean of replicate data and error bars report SD. (A) Left: Timetrace thermal shift curves of BAX WT titrated with 2t–16 (0.02–40 μM) and subjected to MST. Right: Thermophoresis and temperature jump value for BAX WT treated with a range of 2t–16 concentrations fitted to determine a K D value. (B) BAX WT was treated with 2t–16 or 16CHO (0.04, 1.25, 40 μM) and MST timetrace thermal shift curves were fitted using a one-step exponential function and compared using the decay (K) constants normalized by the untreated BAX curve. Original data in Figure S2C . (C) LC-MS of recombinant BAX WT alone or incubated with 2t–16. Samples were then alkylated with iodoacetamide to identify unmodified cysteine residues and trypsin digested for analysis. Four cysteine-containing peptide fragments were detected. Values denote peptide abundance, calculated as AUC for each peak. (D–I) LUV permeabilization studies with recombinant BAX 2S protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from liposomes. Grey data report LUVs solubilized with 1% CHAPS to measure maximal signal. Data shown are the mean of technical replicates. (D) BAX 2S protein (100 nM) was combined with DMSO vehicle or 2t–16 (6.5–50 μM) followed by addition of LUVs and measured by fluorescent spectroscopy. (E) Same as in D with BIM–BH3 peptide (2.5 μM) added to BAX 2S and 2t–16. (F) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX 2S (100 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figure S2B . (G–H) LUV permeabilization studies as in D–E with BAX 2S (100 nM) and hexadecanal (6.5–50 μM) ± BIM–BH3 peptide (2.5 μM). (I) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX 2S (100 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figure S2C . See also .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation, Liquid Chromatography with Mass Spectroscopy, Fluorescence, Liposomes, Spectroscopy

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated as indicated, and subjected to MST. Timetrace thermal shift curves of BAX WT titrated with 2t–16 or 16CHO (0.04, 1.25, 40 μM) report the mean of replicate data. (B−C) The melting temperature of BAX WT and BAX 2S ± 2t–16 (6.5−50 μM) was measured by thermal shift assay using SYPRO orange and compared. Statistical significance was determined by two-way ANOVA; ns, not significant ( P > 0.05). (D–E) LUV permeabilization studies with recombinant BAX 2S protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from compromised liposomes. Left panels: kinetic fluorescence data; right panels: endpoint data normalized to LUV fluorescence and maximal signal generated by LUVs solubilized with CHAPS detergent (grey data). Data are shown as the mean of technical replicates and error bars report SEM. (D) Data summarized by . LUVs permeabilized by BAX 2S (100 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). (E) Data summarized by . LUVs permeabilized by BAX 2S (100 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM).

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation, Thermal Shift Assay, Fluorescence, Liposomes, Generated

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Illustration of BAX and BAK–BH3 interactions within FLAMBE. The reporter is a fluorescently-labeled BAK–BH3 peptide that exhibits changes in Polarization measurements as it is bound by BAX. Over time, the BAX population binds BAK–BH3 peptides resulting in increased Polarization, which eventually plateaus if the entire population forms heterodimers (dotted line). In conditions that activate BAX, activation-induced intramolecular rearrangements within BAX result in the dissociation of the BAK–BH3 peptide and a concomitant decrease in Polarization over time as the population of unbound BAK–BH3 peptide increases (solid line). (B) Illustration of FLAMBE data parameterization and analysis. Left: Kinetic Polarization data is collected for a treatment causing BAX activation and exhibiting accelerated kinetics of BAK TAMRA dissociation (blue lines, depicted as a titration exhibiting dose-dependent BAX activation). Kinetic data is parameterized by extracting endpoint Polarization (EP) and time-to-maximum peak (Tmax) for each condition. Right: Parameterized data are normalized to BAK TAMRA and BAX controls (grey and black, respectively) and titrations or separate conditions can be visualized as a two-dimensional plot. Generally, conditions exhibiting minimal or robust activation of the BAX population cluster in the upper-right or lower-left regions, respectively. Conditions plotted above BAX (i.e., EP > 1) form stable non-activating complexes with the BAX:BAK TAMRA heterodimer (red region). (C) BAX WT (60 nM) was treated with BIM–BH3 peptide (0.25–2 μM) and subjected to FLAMBE to visualize dose-dependent activation-induced dissociation of BAK TAMRA . BIM–BH3 at a low concentration (0.25 μM, dark blue data) demonstrated a stable, non-activating interaction with the BAX:BAK TAMRA complex and exhibited increased Polarization. (D) BAX WT (60 nM) was treated with 16CHO (2–50 μM), combined with BAK TAMRA (50 nM), and subjected to FLAMBE. (E) Same as in D with BAX 2S (60 nM). (F) Left: BAX 2S (60 nM) was treated with three non-activating concentrations of 2t–16 (green: 4.5 μM; orange: 6.5 μM; red: 10 μM). Parameterization of this data is included in . Right: AUC calculated for each condition was normalized to the BAX and BAK TAMRA controls and reported as a percent change from the vehicle-treated BAX condition. (G) Left: BAX WT (60 nM) was combined with a non-activating concentrations of BIM–BH3 peptide (0.15 μM) and 2t–16 (4.5 μM), followed by BAK TAMRA (50 nM), and subjected to FLAMBE. Middle: Parameterized FLAMBE data including three concentrations of 2t–16 (green: 4.5 μM; orange: 6.5 μM; red: 10 μM) in the absence or presence of BIM–BH3 (circle and square datapoints, respectively). Annotations report the magnitude of shift between data with and without BIM–BH3. Right: AUC calculated for each condition was normalized to the BAX and BAK TAMRA controls and reported as a percent change from the vehicle-treated BAX condition. (H) Fluorescence polarization competition assay with recombinant BCL-xL ΔC protein treated with 2t–16 (2–50 μM) and combined with BAK TAMRA .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Activation Assay, Titration, Concentration Assay, Fluorescence, Competitive Binding Assay, Recombinant

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated with the indicated 2t–alkenals (0.16–5 μM), and subjected to MST. Timetrace data are shown as the mean of replicates. Thermophoresis metrics for each 2t–alkenal are summarized in . (B) BAX 2S (60 nM) was treated with the indicated 2t–alkenal (3–50 μM), combined with BAK TAMRA (50 nM), and subjected to FLAMBE. Data are shown as the mean of replicates. Parameterized data reporting EP and Tmax for each experiment are provided in Figures 5D –E . (C) LUVs permeabilized by BAX 2S (100 nM) treated with the indicated 2t–alkenal (6.5–50 μM). Data are shown as the mean of replicates. Normalized endpoint permeabilization data summarized in .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation