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: Antibodies for immunoblotting: mouse monoclonal anti-eIF4E (cat# 610270, BD Biosciences), mouse monoclonal anti-β-Actin (cat# A5441, Sigma Aldrich), rabbit polyclonal anti-Mcl-1 (S-19) (cat# sc-819, Santa Cruz), mouse monoclonal anti-HSP90α/β (F-8) (cat# sc-13119, Santa Cruz), rabbit polyclonal anti-Myc (cat# ab32072, Abcam), Mouse monoclonal anti-CD44 antibody (cat# 156–3 C11, Cell Signaling Technology), rabbit polyclonal anti-CD44 (cat# A12410, Abclonal), rabbit polyclonal anti-HAS3 antibody (cat# ab154104, Abcam), rabbit polyclonal anti-phosphoglucomutase 5 (cat# AI14638, Abgent), rabbit polyclonal anti-Lamin A (C-terminal) (cat# L1293, Sigma Aldrich), rabbit polyclonal anti-GAPDH (FL-335) (cat# sc-25778, Santa Cruz), rabbit polyclonal anti-UGDH (cat#AP12613b-EV, Abgent), rabbit monoclonal anti-Hexokinase II (cat# 2867, Cell Signaling Technology), rabbit monoclonal anti-UAP1 antibody (cat# 2716, GenuinBiotech), mouse monoclonal anti-RPL17 (C-8, cat# sc-515904, Santa Cruz), mouse monoclonal anti-RPS16 (D-8, cat# sc-518206; Santa Cruz), mouse monoclonal anti-RPS6 (C-8, cat# sc-74459, Santa Cruz), mouse monoclonal anti-eIF3e (G-7, cat# sc-390413, Santa Cruz), mouse monoclonal anti-eIF3p110 (B-6, cat# sc-74507, Santa Cruz), mouse monoclonal anti-eIF3b (A-7, cat# sc-374156, Santa Cruz), mouse monoclonal anti-eIF4AI/II (H-5, cat# sc-377315, Santa Cruz), mouse monoclonal anti-eIF4G (A-10, cat# sc-133155, Santa Cruz), mouse monoclonal anti-Nopp140 (E-7, cat# sc-374033, Santa Cruz), rabbit monoclonal anti-histone H3 acetyl K27 (cat# ab177178, Abcam), rabbit polyclonal Calreticulin (cat# AW5211, ABCEPTA), rabbit polyclonal anti-Calnexin (cat# ab22595, Abcam), rabbit polyclonal anti-Ezrin antibody (cat# PA5-80603, Invitrogen), mouse monoclonal anti-Ezrin (CPTC-Ezrin-1, cat# AB_2100318, DSHB), mouse monoclonal anti-MEK-1 (H-8, sc-6250, Santa Cruz), and mouse monoclonal anti-cytochrome c (A-8, cat# sc-13156, Santa Cruz).
Techniques: Western Blot, Suspension, Knockdown, Luciferase, Negative Control, Control, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Fractionation, Immunofluorescence, Confocal Microscopy
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![A , Western blot showing indicated proteins expression levels in YUMM1.1 cells expressing a shRNA control or targeting ASS1. HSP90 serves as a loading control. B , Western blot showing indicated proteins expression levels in YUMM1.7 cells expressing a shRNA control or targeting ASS1. HSP90 serves as a loading control. C , Western blot showing indicated proteins expression levels in YUMM2.1 cells expressing an ectopic empty vector or containing the open reading frame of ASS1. HSP90 serves as a loading control. D , Renilla over Firefly luminescent ratio quantification in YUMM1.1 cells expressing a shRNA control or targeting ASS1 (n=4; mean ± SEM). E , Renilla over Firefly luminescent ratio quantification in YUMM1.7 cells expressing a shRNA control or targeting ASS1 (n=3; mean ± SEM). F , Renilla over Firefly luminescent ratio quantification in YUMM2.1 cells expressing an ectopic empty vector or containing the open reading frame of ASS1 (n=3; mean ± SEM). G , Protein synthesis rates were determined in YUMM1.1 cells expressing a shRNA control or targeting ASS1. Cells were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 4). H , Protein synthesis rates were determined in YUMM1.7 cells expressing a shRNA control or targeting ASS1. Cells were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). I , Protein synthesis rates were determined in YUMM2.1 cells expressing an ectopic empty vector or containing the open reading frame of ASS1. Cells were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). J , Proximity Ligation Assay showing eiF4E/eiF4G interactions in YUMM1.1 cells expressing a shRNA control or targeting ASS1. Representative images are presented in the left panel, and quantifications are presented in the right panel. K , Proximity Ligation Assay showing eiF4E/eiF4G interactions in YUMM1.7 cells expressing a shRNA control or targeting ASS1. Representative images are presented in the left panel, and quantifications are presented in the right panel.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_79/10__64898_slash_2026__03__17__712479/10__64898_slash_2026__03__17__712479___F3.large.jpg)