mouse anti alpha tubulin hybridoma supernatant  (Developmental Studies Hybridoma Bank)

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: IL-18 metabolically reprograms CAR-expressing natural killer T cells and enhances their antitumor activity

doi: 10.1016/j.ymthe.2026.01.001

Figure Lengend Snippet: A. Flow cytometry histograms depicting MitoTracker Green (MG, left) and tetramethylrhodamine ethyl ester perchlorate (TMRE, right) staining in CAR + NKTs from a representative donor 3 days after tumor stimulation. B. Summary data for MG and TMRE staining after tumor stimulation (left; mean, n =12 donors, three independent experiments) and TMRE-to-MG ratio calculated from values in left panel (right; mean, n =12 donors, three independent experiments), * p < 0.05, ** p < 0.01, **** p < 0.0001, paired two-tailed t-test. C. Differentially expressed metabolites in iC9.IL18 versus iC9.IL15 expressing CAR-NKTs 24 hours after stimulation with plate-bound 1A7 antibody ( n =4 donors, p <0.05). D. Heatmap depicting oxidative phosphorylation metabolite related genes in CAR-NKTs after one stimulation (left). TCA cycle schematic showing significantly upregulated molecules in red text. Purple italic text indicates upregulated transcripts with p <0.05 and adjusted p >0.05 (right). E. Heatmap depicting glycolysis metabolite-related genes in CAR-NKTs after one stimulation (left). Glycolysis pathway schematic showing significantly upregulated molecules in red text. Purple italic text indicates upregulated transcripts with p <0.05 and adjusted p >0.05 (right). F. Gene set enrichment analysis of oxidative phosphorylation (yellow) and glycolysis signatures (blue) in stimulated CAR-NKTs expressing iC9.IL18 versus iC9.IL15.

Article Snippet: GD2-CAR expression was evaluated using an AF647 14G2a anti-idiotype 1A7 antibody purified from an 1A7 mouse hybridoma (ATCC, HB-11786), which was custom-conjugated to Alexa Fluor 647 by BioLegend.

Techniques: Expressing, Flow Cytometry, Staining, Two Tailed Test, Phospho-proteomics

Journal: bioRxiv

Article Title: eIF4E and Ezrin cooperate in pseudopods to drive a localized migratory translation program in acute myeloid leukemia

doi: 10.64898/2026.02.21.707190

Figure Lengend Snippet: A. Western blot of total cell lysates from MM6 cells grown in suspension demonstrated knockdown of Ezrin (siEZR) or eIF4E (siEIF4E) compared to RNAi to luciferase (siLUC) used as a negative control. b-Actin is provided as a loading control. Other proteins of the Ezrin-CD44-HA axis are also shown. Quantification for these is shown in with 3-6 biological replicates for each protein. B. Adhesion and invasion capacity of MM6 cells onto/through HS-5 bone marrow stroma. Fold change relative to siLUC is shown. Each symbol represents a biological replicate performed independently with replicates. Bars represent the mean, shown with standard deviations and p-values (one-way ANOVA). C. Western blot of eIF4E and Ezrin immunoprecipitations (IPs) using total cell lysates from MM6 cells in suspension. SN, supernatant after immunoprecipitation, IgG, negative control. Representative of three biological replicates. IPs of total cell lysates from THP-1 cells in suspension are provided in . D. IPs from MM6 cells in suspension using the rRNA antibody Y10b. LC indicates antibody light chain. Representative of three biological replicates. E. RNA immunoprecipitations (RIPs) from MM6 total cell lysates grown in suspension using anti-Ezrin (Ezrin RIP) or anti-eIF4E (eIF4E RIP) antibodies. Data are from RT-qPCR and represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). F. Western blots of eIF4E and Ezrin IPs from the cytoplasmic fractions of MM6 cells in suspension or G . After invasion through HS-5 bone marrow stroma (invaded). Fractionation controls are provided in . H. RIPs from MM6 cytoplasmic fraction from invaded cells using anti-eIF4E (eIF4E RIP) or anti-Ezrin (Ezrin RIP) antibodies. Data are from RT-qPCR represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). I. Count of the number of pseudopods observed in suspension and invaded MM6 cells represented as a fraction relative to the total cells counted. Each symbol represents a biological replicate. Bars represent the mean, shown with standard deviations and p-values (two-way ANOVA). J . Immunofluorescence and confocal microscopy demonstrating eIF4E, Ezrin, CD44 and rRNA are localized to the same pseudopods (white arrows). All confocal micrographs represent a single section through the plane of the cell. Scale bar = 10 µm

Article Snippet: In case of Y10b IPs, 100 ml of Y10b mouse hybridoma supernatant (DSHB), or 10 mg od mouse IgG (cat# 12-371, Millipore) were bound to the beads 50 μL protein G conjugated superparamagnetic beads in 1 ml of NT2 buffer ON at 4°C, washed three times with NT2 buffer, added to 1 mg of total cell lysates, incubated overnight at 4°C with rotation and washed as described for IPs with rabbit antibodies.

Techniques: Western Blot, Suspension, Knockdown, Luciferase, Negative Control, Control, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Fractionation, Immunofluorescence, Confocal Microscopy