Review





Similar Products

mouse monoclonal anti ezrin  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank mouse monoclonal anti ezrin
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 <t>using</t> <t>anti-Ezrin</t> (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
Mouse Monoclonal Anti Ezrin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ezrin/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
mouse monoclonal anti ezrin - by Bioz Stars, 2026-04
94/100 stars
  Buy from Supplier
ezrin antibody / ezr  (NSJ Bioreagents)
91
NSJ Bioreagents ezrin antibody / ezr
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 <t>using</t> <t>anti-Ezrin</t> (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
Ezrin Antibody / Ezr, supplied by NSJ Bioreagents, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ezrin antibody / ezr/product/NSJ Bioreagents
Average 91 stars, based on 1 article reviews
ezrin antibody / ezr - by Bioz Stars, 2026-04
91/100 stars
  Buy from Supplier
ezrin  (Addgene inc)
96
Addgene inc ezrin
(A) Measurements <t>of</t> <t>NHE1</t> activity at prescribed pH e in the absence or presence of EIPA (40 μM) using NH 4 Cl prepulse technique by quantifying the pH i recovery of pHrodo-loaded cells. (B) ΔpH/min values were obtained after removal of NH 4 Cl medium and calculated by linear regression fitting of the initial recovery phase. Data represent mean ± SD of the individual averages from three experiments for n ≥ 51 cells. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (C) Immunofluorescence images of cells in confinement at pH e = 7.4 or 6.4, and stained for NHE1 (red), <t>ezrin</t> (green), and Hoechst (blue). (D) Percentage of cells displaying NHE1 and ezrin co-polarization at prescribed pH e . Data represent mean ± SD from three experiments. ** p < 0.01 by unpaired t test. (E) Western blot images and quantification of NHE1 expression. Data represent mean ± SD from four experiments. * p < 0.05 by unpaired t test. (F) Representative PLA images of NHE1-pAkt interaction after scramble control (shControl) or shNHE1 cell preconditioning at prescribed pH e for 24 h. (G) Quantification of PLA interaction normalized to total number of dots per nuclei. Data represent mean ± SD of multiple fields from four experiments. ** p < 0.01 and **** p < 0.0001 by one-way ANOVA followed by Tukey’s test after log transformation. (H and J) Western blot images of pAkt and tAkt (H), and NHE1 (J) before and after pH e change from 7.4 to 6.4 at prescribed time points (0.5, 1, and 2 h). (I) Quantification of pAkt and tAkt normalized by GAPDH (left y axis) and of pAkt normalized by tAkt (right y axis) from five experiments. * p < 0.05 and ** p < 0.01 by Kruskal-Wallis followed by Dunn’s. (K) Quantification of NHE1 expression from four experiments. (L) Western blot images of pAkt and tAkt at pH e of 7.4 before and after EIPA (40 μM) treatment for prescribed time points (0.5, 1, and 2 h). (M) Quantification of pAkt and tAkt, as described in (I), from three experiments before and after the addition of EIPA (40 μM). * p < 0.05 and ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (N) Western blot images of pAkt and tAkt for SC and NHE1-KD cells. (O) Quantification of pAkt and tAkt normalized, as described in (I), from five experiments. * p < 0.05, ** p < 0.01 by unpaired t test. Data represent mean ± SD. Cell model: MDA-MB-231.
Ezrin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ezrin/product/Addgene inc
Average 96 stars, based on 1 article reviews
ezrin - by Bioz Stars, 2026-04
96/100 stars
  Buy from Supplier
rabbit polyclonal anti ezrin antibody  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank rabbit polyclonal anti ezrin antibody
A. Representative western blot from total cell lysates of MM6 and NOMO-1 cell lines demonstrating NOMO-1 cells have similar eIF4E levels to CD34+ from healthy donors, while MM6 cells have similar levels to high-eIF4E AML patient specimens. b-Actin is provided for loading control. Each lane refers to a different sample. B . Confocal micrograph of eIF4E nuclear and cytoplasmic staining in MM6 cells. Scale bar = 10 µm. C, D. Quantitation of western blots for NOMO-1 eIF4E relative to vector or MM6 CRISPR 4E relative to CRISPR-CTRL cells ( , Supplemental Figure 1G). Each data point represents a biological replicate. Bar represents the mean. Standard deviation and p-values (Welch’s t test) are shown. E. Representative confocal micrograph in NOMO-1 eIF4E cells demonstrating that HA staining is specific as its signal is removed upon treatment with hyaluronidase (HAse). HA is red; DAPI is blue. F. Representative western blot of MM6 cells treated with the eIF4E inhibitor ribavirin or vehicle control demonstrating that ribavirin reduces eIF4E target proteins including <t>Ezrin.</t> b-Actin is provided as a loading control. G, H . Representative western blots demonstrating lower eIF4E levels and factors in the Ezrin-CD44-HA axis in MM6 and THP-1 CRISPR-4E cells compared to CRISPR-Controls.
Rabbit Polyclonal Anti Ezrin Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal anti ezrin antibody/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
rabbit polyclonal anti ezrin antibody - by Bioz Stars, 2026-04
94/100 stars
  Buy from Supplier
ezrin / p81(cptc-ezrin-1)  (Biotium)
96
Biotium ezrin / p81(cptc-ezrin-1)
A. Representative western blot from total cell lysates of MM6 and NOMO-1 cell lines demonstrating NOMO-1 cells have similar eIF4E levels to CD34+ from healthy donors, while MM6 cells have similar levels to high-eIF4E AML patient specimens. b-Actin is provided for loading control. Each lane refers to a different sample. B . Confocal micrograph of eIF4E nuclear and cytoplasmic staining in MM6 cells. Scale bar = 10 µm. C, D. Quantitation of western blots for NOMO-1 eIF4E relative to vector or MM6 CRISPR 4E relative to CRISPR-CTRL cells ( , Supplemental Figure 1G). Each data point represents a biological replicate. Bar represents the mean. Standard deviation and p-values (Welch’s t test) are shown. E. Representative confocal micrograph in NOMO-1 eIF4E cells demonstrating that HA staining is specific as its signal is removed upon treatment with hyaluronidase (HAse). HA is red; DAPI is blue. F. Representative western blot of MM6 cells treated with the eIF4E inhibitor ribavirin or vehicle control demonstrating that ribavirin reduces eIF4E target proteins including <t>Ezrin.</t> b-Actin is provided as a loading control. G, H . Representative western blots demonstrating lower eIF4E levels and factors in the Ezrin-CD44-HA axis in MM6 and THP-1 CRISPR-4E cells compared to CRISPR-Controls.
Ezrin / P81(Cptc Ezrin 1), supplied by Biotium, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ezrin / p81(cptc-ezrin-1)/product/Biotium
Average 96 stars, based on 1 article reviews
ezrin / p81(cptc-ezrin-1) - by Bioz Stars, 2026-04
96/100 stars
  Buy from Supplier
anti ezrin  (Proteintech)
93
Proteintech anti ezrin
A. Representative western blot from total cell lysates of MM6 and NOMO-1 cell lines demonstrating NOMO-1 cells have similar eIF4E levels to CD34+ from healthy donors, while MM6 cells have similar levels to high-eIF4E AML patient specimens. b-Actin is provided for loading control. Each lane refers to a different sample. B . Confocal micrograph of eIF4E nuclear and cytoplasmic staining in MM6 cells. Scale bar = 10 µm. C, D. Quantitation of western blots for NOMO-1 eIF4E relative to vector or MM6 CRISPR 4E relative to CRISPR-CTRL cells ( , Supplemental Figure 1G). Each data point represents a biological replicate. Bar represents the mean. Standard deviation and p-values (Welch’s t test) are shown. E. Representative confocal micrograph in NOMO-1 eIF4E cells demonstrating that HA staining is specific as its signal is removed upon treatment with hyaluronidase (HAse). HA is red; DAPI is blue. F. Representative western blot of MM6 cells treated with the eIF4E inhibitor ribavirin or vehicle control demonstrating that ribavirin reduces eIF4E target proteins including <t>Ezrin.</t> b-Actin is provided as a loading control. G, H . Representative western blots demonstrating lower eIF4E levels and factors in the Ezrin-CD44-HA axis in MM6 and THP-1 CRISPR-4E cells compared to CRISPR-Controls.
Anti Ezrin, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti ezrin/product/Proteintech
Average 93 stars, based on 1 article reviews
anti ezrin - by Bioz Stars, 2026-04
93/100 stars
  Buy from Supplier
anti phospho ezrin thr567  (Proteintech)
93
Proteintech anti phospho ezrin thr567
A. Representative western blot from total cell lysates of MM6 and NOMO-1 cell lines demonstrating NOMO-1 cells have similar eIF4E levels to CD34+ from healthy donors, while MM6 cells have similar levels to high-eIF4E AML patient specimens. b-Actin is provided for loading control. Each lane refers to a different sample. B . Confocal micrograph of eIF4E nuclear and cytoplasmic staining in MM6 cells. Scale bar = 10 µm. C, D. Quantitation of western blots for NOMO-1 eIF4E relative to vector or MM6 CRISPR 4E relative to CRISPR-CTRL cells ( , Supplemental Figure 1G). Each data point represents a biological replicate. Bar represents the mean. Standard deviation and p-values (Welch’s t test) are shown. E. Representative confocal micrograph in NOMO-1 eIF4E cells demonstrating that HA staining is specific as its signal is removed upon treatment with hyaluronidase (HAse). HA is red; DAPI is blue. F. Representative western blot of MM6 cells treated with the eIF4E inhibitor ribavirin or vehicle control demonstrating that ribavirin reduces eIF4E target proteins including <t>Ezrin.</t> b-Actin is provided as a loading control. G, H . Representative western blots demonstrating lower eIF4E levels and factors in the Ezrin-CD44-HA axis in MM6 and THP-1 CRISPR-4E cells compared to CRISPR-Controls.
Anti Phospho Ezrin Thr567, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti phospho ezrin thr567/product/Proteintech
Average 93 stars, based on 1 article reviews
anti phospho ezrin thr567 - by Bioz Stars, 2026-04
93/100 stars
  Buy from Supplier
ezrin mouse monoclonal antibody  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank ezrin mouse monoclonal antibody
a) <t>Ezrin</t> protein sequence: membrane binding FERM domain consists of three lobes – F1 (residues P2-P86), F2 (residues E87-G202), and F3 (residues I203-K296); linker region consists of residues P297-P496; and the actin-binding C-terminal domain (CTD) consists of residues T497-L586. b) AlphaFold 3.0 predicted closed-form structure of the full-length ezrin protein . In the predicted closed-form ezrin structure, C-terminal CTD domain interacts with FERM over the F2-F3 subdomain surface. c) System setup for the FERM-CTD structure (PDB ID: 4RM9) at a DOPC:DOPS:PIP 2 (80:16:4ratio) membrane that was used in the molecular dynamics simulations in this study. d) Number of PIP 2 head groups phosphorus atoms within 10 Å of any atoms in the residues in FERM. Asterisks (*) indicate significant difference (α = 0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns, e) Aggregation of PIP 2 (orange), DOPS (blue) and DOPC (gray) phospholipids in a PIP 2 /DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark. f) Number of DOPS within 10 Å of the residues in FERM. Asterisks (*) indicate significant difference (independent sample t-test, α=0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns. Double asterisk (**) indicates significant difference (ANOVA, α = 0.05) between the means in systems with DOPC/DOPS/PIP 2 membranes and DOPC/DOPS membranes in the 990-1000 ns interval. g) Aggregation of DOPS phospholipids (blue) in a DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark.
Ezrin Mouse Monoclonal Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ezrin mouse monoclonal antibody/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
ezrin mouse monoclonal antibody - by Bioz Stars, 2026-04
94/100 stars
  Buy from Supplier
pegfp n1 ezrin human  (Addgene inc)
94
Addgene inc pegfp n1 ezrin human
a) <t>Ezrin</t> protein sequence: membrane binding FERM domain consists of three lobes – F1 (residues P2-P86), F2 (residues E87-G202), and F3 (residues I203-K296); linker region consists of residues P297-P496; and the actin-binding C-terminal domain (CTD) consists of residues T497-L586. b) AlphaFold 3.0 predicted closed-form structure of the full-length ezrin protein . In the predicted closed-form ezrin structure, C-terminal CTD domain interacts with FERM over the F2-F3 subdomain surface. c) System setup for the FERM-CTD structure (PDB ID: 4RM9) at a DOPC:DOPS:PIP 2 (80:16:4ratio) membrane that was used in the molecular dynamics simulations in this study. d) Number of PIP 2 head groups phosphorus atoms within 10 Å of any atoms in the residues in FERM. Asterisks (*) indicate significant difference (α = 0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns, e) Aggregation of PIP 2 (orange), DOPS (blue) and DOPC (gray) phospholipids in a PIP 2 /DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark. f) Number of DOPS within 10 Å of the residues in FERM. Asterisks (*) indicate significant difference (independent sample t-test, α=0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns. Double asterisk (**) indicates significant difference (ANOVA, α = 0.05) between the means in systems with DOPC/DOPS/PIP 2 membranes and DOPC/DOPS membranes in the 990-1000 ns interval. g) Aggregation of DOPS phospholipids (blue) in a DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark.
Pegfp N1 Ezrin Human, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pegfp n1 ezrin human/product/Addgene inc
Average 94 stars, based on 1 article reviews
pegfp n1 ezrin human - by Bioz Stars, 2026-04
94/100 stars
  Buy from Supplier

Image Search Results


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

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

(A) Measurements of NHE1 activity at prescribed pH e in the absence or presence of EIPA (40 μM) using NH 4 Cl prepulse technique by quantifying the pH i recovery of pHrodo-loaded cells. (B) ΔpH/min values were obtained after removal of NH 4 Cl medium and calculated by linear regression fitting of the initial recovery phase. Data represent mean ± SD of the individual averages from three experiments for n ≥ 51 cells. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (C) Immunofluorescence images of cells in confinement at pH e = 7.4 or 6.4, and stained for NHE1 (red), ezrin (green), and Hoechst (blue). (D) Percentage of cells displaying NHE1 and ezrin co-polarization at prescribed pH e . Data represent mean ± SD from three experiments. ** p < 0.01 by unpaired t test. (E) Western blot images and quantification of NHE1 expression. Data represent mean ± SD from four experiments. * p < 0.05 by unpaired t test. (F) Representative PLA images of NHE1-pAkt interaction after scramble control (shControl) or shNHE1 cell preconditioning at prescribed pH e for 24 h. (G) Quantification of PLA interaction normalized to total number of dots per nuclei. Data represent mean ± SD of multiple fields from four experiments. ** p < 0.01 and **** p < 0.0001 by one-way ANOVA followed by Tukey’s test after log transformation. (H and J) Western blot images of pAkt and tAkt (H), and NHE1 (J) before and after pH e change from 7.4 to 6.4 at prescribed time points (0.5, 1, and 2 h). (I) Quantification of pAkt and tAkt normalized by GAPDH (left y axis) and of pAkt normalized by tAkt (right y axis) from five experiments. * p < 0.05 and ** p < 0.01 by Kruskal-Wallis followed by Dunn’s. (K) Quantification of NHE1 expression from four experiments. (L) Western blot images of pAkt and tAkt at pH e of 7.4 before and after EIPA (40 μM) treatment for prescribed time points (0.5, 1, and 2 h). (M) Quantification of pAkt and tAkt, as described in (I), from three experiments before and after the addition of EIPA (40 μM). * p < 0.05 and ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (N) Western blot images of pAkt and tAkt for SC and NHE1-KD cells. (O) Quantification of pAkt and tAkt normalized, as described in (I), from five experiments. * p < 0.05, ** p < 0.01 by unpaired t test. Data represent mean ± SD. Cell model: MDA-MB-231.

Journal: Cell reports

Article Title: Hypoxia restores the acidosis-induced inhibition of cancer cell dissemination

doi: 10.1016/j.celrep.2026.116970

Figure Lengend Snippet: (A) Measurements of NHE1 activity at prescribed pH e in the absence or presence of EIPA (40 μM) using NH 4 Cl prepulse technique by quantifying the pH i recovery of pHrodo-loaded cells. (B) ΔpH/min values were obtained after removal of NH 4 Cl medium and calculated by linear regression fitting of the initial recovery phase. Data represent mean ± SD of the individual averages from three experiments for n ≥ 51 cells. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (C) Immunofluorescence images of cells in confinement at pH e = 7.4 or 6.4, and stained for NHE1 (red), ezrin (green), and Hoechst (blue). (D) Percentage of cells displaying NHE1 and ezrin co-polarization at prescribed pH e . Data represent mean ± SD from three experiments. ** p < 0.01 by unpaired t test. (E) Western blot images and quantification of NHE1 expression. Data represent mean ± SD from four experiments. * p < 0.05 by unpaired t test. (F) Representative PLA images of NHE1-pAkt interaction after scramble control (shControl) or shNHE1 cell preconditioning at prescribed pH e for 24 h. (G) Quantification of PLA interaction normalized to total number of dots per nuclei. Data represent mean ± SD of multiple fields from four experiments. ** p < 0.01 and **** p < 0.0001 by one-way ANOVA followed by Tukey’s test after log transformation. (H and J) Western blot images of pAkt and tAkt (H), and NHE1 (J) before and after pH e change from 7.4 to 6.4 at prescribed time points (0.5, 1, and 2 h). (I) Quantification of pAkt and tAkt normalized by GAPDH (left y axis) and of pAkt normalized by tAkt (right y axis) from five experiments. * p < 0.05 and ** p < 0.01 by Kruskal-Wallis followed by Dunn’s. (K) Quantification of NHE1 expression from four experiments. (L) Western blot images of pAkt and tAkt at pH e of 7.4 before and after EIPA (40 μM) treatment for prescribed time points (0.5, 1, and 2 h). (M) Quantification of pAkt and tAkt, as described in (I), from three experiments before and after the addition of EIPA (40 μM). * p < 0.05 and ** p < 0.01 by one-way ANOVA followed by Tukey’s test. (N) Western blot images of pAkt and tAkt for SC and NHE1-KD cells. (O) Quantification of pAkt and tAkt normalized, as described in (I), from five experiments. * p < 0.05, ** p < 0.01 by unpaired t test. Data represent mean ± SD. Cell model: MDA-MB-231.

Article Snippet: Lentiviral shRNAs targeting NHE1 and ezrin were generated by subcloning the following sequences into pLKO.1 puro (plasmid #8453, Addgene) as previously described., non-targeting scramble control: 5′ -GCACTACCAGAGCTAACTCAGATAGTACT-3’. human shEZRIN: 5′ -CCGTGGGATGCTCAAAGATAA-3’. human sh1NHE1: 5′ -GACAAGCTCAACCGGTTTAAT-3’. human sh2NHE1: 5′ -CCAATCTTAGTTTCTAACCAA-3’.

Techniques: Activity Assay, Immunofluorescence, Staining, Western Blot, Expressing, Control, Transformation Assay

(A–D) Western blot images (A and C) and quantification of ILK expression (B and D) in WT (B), SC and NHE1-KD (D) cells following preconditioning at pH e = 7.4 or 6.4 for 24 h. Data represent mean ± SD from N = 3 experiments. * p < 0.05 and ** p < 0.01 by unpaired t test (B) or by one-way ANOVA followed by Tukey’s test (D). (E–H) Western blot images (E and G) and quantification of ILK expression in cells preconditioned at pH e of 7.4 or 6.4 for 24 h in the presence of LY294002 (LY; 10 μM, 24 h) (E and F), verteporfin (Vert., 0.3 μM, 24 h) (G and H), or vehicle control. Data represent mean ± SD from N = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Tukey’s test. (I) Immunofluorescence staining of cells with NHE1 (magenta), ILK (white), and Hoechst (blue) following their exposure to pH e = 7.4 or 6.4 for 24 h. (J) Immunofluorescence staining with ezrin (green), ILK (white), β1-integrin (red), and Hoechst (blue) following their exposure to pH e = 7.4 or 6.4 for 24 h. (K) SC or NHE1-KD cell migration velocities on 2D following their preconditioning at prescribed pH e for 24 h in the presence of either the ILK inhibitor CPD22 (2.5 μM) or vehicle control (VC). Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 17 cells. * p < 0.05 by one-way ANOVA followed by Tukey’s test. (L) Representative phase contrast images of cells dissociating from 3D breast cancer spheroids embedded in 3D collagen gels at the indicated time points in the presence of either CPD22 (2.5 μM) or VC, following cell preconditioning at pH e 7.4 or 6.4 for 24 h. (M) Number of dissociated cells from 3D spheroids embedded in 3D collagen gels at t = 22 h in the presence of CPD22 (2.5 μM) or VC at prescribed pH e . Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 7 spheroids. * p < 0.05 by one-way ANOVA followed by Tukey’s test. Cell model: MDA-MB-231. Scale bars: 10 μm (I and J) or 100 μm (L).

Journal: Cell reports

Article Title: Hypoxia restores the acidosis-induced inhibition of cancer cell dissemination

doi: 10.1016/j.celrep.2026.116970

Figure Lengend Snippet: (A–D) Western blot images (A and C) and quantification of ILK expression (B and D) in WT (B), SC and NHE1-KD (D) cells following preconditioning at pH e = 7.4 or 6.4 for 24 h. Data represent mean ± SD from N = 3 experiments. * p < 0.05 and ** p < 0.01 by unpaired t test (B) or by one-way ANOVA followed by Tukey’s test (D). (E–H) Western blot images (E and G) and quantification of ILK expression in cells preconditioned at pH e of 7.4 or 6.4 for 24 h in the presence of LY294002 (LY; 10 μM, 24 h) (E and F), verteporfin (Vert., 0.3 μM, 24 h) (G and H), or vehicle control. Data represent mean ± SD from N = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Tukey’s test. (I) Immunofluorescence staining of cells with NHE1 (magenta), ILK (white), and Hoechst (blue) following their exposure to pH e = 7.4 or 6.4 for 24 h. (J) Immunofluorescence staining with ezrin (green), ILK (white), β1-integrin (red), and Hoechst (blue) following their exposure to pH e = 7.4 or 6.4 for 24 h. (K) SC or NHE1-KD cell migration velocities on 2D following their preconditioning at prescribed pH e for 24 h in the presence of either the ILK inhibitor CPD22 (2.5 μM) or vehicle control (VC). Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 17 cells. * p < 0.05 by one-way ANOVA followed by Tukey’s test. (L) Representative phase contrast images of cells dissociating from 3D breast cancer spheroids embedded in 3D collagen gels at the indicated time points in the presence of either CPD22 (2.5 μM) or VC, following cell preconditioning at pH e 7.4 or 6.4 for 24 h. (M) Number of dissociated cells from 3D spheroids embedded in 3D collagen gels at t = 22 h in the presence of CPD22 (2.5 μM) or VC at prescribed pH e . Data represent mean ± SD of the average value of each biological repeat ( N = 3) each with n ≥ 7 spheroids. * p < 0.05 by one-way ANOVA followed by Tukey’s test. Cell model: MDA-MB-231. Scale bars: 10 μm (I and J) or 100 μm (L).

Article Snippet: Lentiviral shRNAs targeting NHE1 and ezrin were generated by subcloning the following sequences into pLKO.1 puro (plasmid #8453, Addgene) as previously described., non-targeting scramble control: 5′ -GCACTACCAGAGCTAACTCAGATAGTACT-3’. human shEZRIN: 5′ -CCGTGGGATGCTCAAAGATAA-3’. human sh1NHE1: 5′ -GACAAGCTCAACCGGTTTAAT-3’. human sh2NHE1: 5′ -CCAATCTTAGTTTCTAACCAA-3’.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Migration

A. Representative western blot from total cell lysates of MM6 and NOMO-1 cell lines demonstrating NOMO-1 cells have similar eIF4E levels to CD34+ from healthy donors, while MM6 cells have similar levels to high-eIF4E AML patient specimens. b-Actin is provided for loading control. Each lane refers to a different sample. B . Confocal micrograph of eIF4E nuclear and cytoplasmic staining in MM6 cells. Scale bar = 10 µm. C, D. Quantitation of western blots for NOMO-1 eIF4E relative to vector or MM6 CRISPR 4E relative to CRISPR-CTRL cells ( , Supplemental Figure 1G). Each data point represents a biological replicate. Bar represents the mean. Standard deviation and p-values (Welch’s t test) are shown. E. Representative confocal micrograph in NOMO-1 eIF4E cells demonstrating that HA staining is specific as its signal is removed upon treatment with hyaluronidase (HAse). HA is red; DAPI is blue. F. Representative western blot of MM6 cells treated with the eIF4E inhibitor ribavirin or vehicle control demonstrating that ribavirin reduces eIF4E target proteins including Ezrin. b-Actin is provided as a loading control. G, H . Representative western blots demonstrating lower eIF4E levels and factors in the Ezrin-CD44-HA axis in MM6 and THP-1 CRISPR-4E cells compared to CRISPR-Controls.

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. Representative western blot from total cell lysates of MM6 and NOMO-1 cell lines demonstrating NOMO-1 cells have similar eIF4E levels to CD34+ from healthy donors, while MM6 cells have similar levels to high-eIF4E AML patient specimens. b-Actin is provided for loading control. Each lane refers to a different sample. B . Confocal micrograph of eIF4E nuclear and cytoplasmic staining in MM6 cells. Scale bar = 10 µm. C, D. Quantitation of western blots for NOMO-1 eIF4E relative to vector or MM6 CRISPR 4E relative to CRISPR-CTRL cells ( , Supplemental Figure 1G). Each data point represents a biological replicate. Bar represents the mean. Standard deviation and p-values (Welch’s t test) are shown. E. Representative confocal micrograph in NOMO-1 eIF4E cells demonstrating that HA staining is specific as its signal is removed upon treatment with hyaluronidase (HAse). HA is red; DAPI is blue. F. Representative western blot of MM6 cells treated with the eIF4E inhibitor ribavirin or vehicle control demonstrating that ribavirin reduces eIF4E target proteins including Ezrin. b-Actin is provided as a loading control. G, H . Representative western blots demonstrating lower eIF4E levels and factors in the Ezrin-CD44-HA axis in MM6 and THP-1 CRISPR-4E cells compared to CRISPR-Controls.

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, Control, Staining, Quantitation Assay, Plasmid Preparation, CRISPR, Standard Deviation

B. The impact on the Ezrin-CD44-HA axis of NOMO-1 eIF4E overexpression (eIF4E) compared to vector control (A) or of MM6 CRISPR-4E cells relative to MM6 CRISPR-CTRL cells (B). Left panels. Western blots of eIF4E and Ezrin-CD44-HA axis. Corresponding b-Actin shown for loading controls. Right panels . Confocal micrographs (single section through the plane of cells) of the indicated cells stained for HA or CD44 shown in red. DAPI shown in blue. Scale bar = 10µm. C. Western blot of primary specimens from AML patients (with normal or high-eIF4E) and CD34 + from healthy donors showing expression of eIF4E and Ezrin. b-Actin is provided as a loading control. Each lane represents a different individual. D. Adhesion (left) and invasion (right) capacity using HS-5 stromal cells of NOMO-1 and MM6 cell lines as a function of genetic eIF4E overexpression or CRISPR knockdown respectively. Data is presented as fold change relative to their corresponding controls. Each symbol represents an independent experiment performed with replicates. The bar represents the mean with standard deviations and p-values calculated with two tailed paired T test. E. Schematic representation of colonization assay into mesenchymal stromal cell spheroid model mimicking the bone marrow niche. F. Quantification of AML cell colonization capacity relative to their corresponding controls. Each symbol represents an experimental replicate. The bar represents the mean with standard deviations and p-values, Welch’s t test.

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: B. The impact on the Ezrin-CD44-HA axis of NOMO-1 eIF4E overexpression (eIF4E) compared to vector control (A) or of MM6 CRISPR-4E cells relative to MM6 CRISPR-CTRL cells (B). Left panels. Western blots of eIF4E and Ezrin-CD44-HA axis. Corresponding b-Actin shown for loading controls. Right panels . Confocal micrographs (single section through the plane of cells) of the indicated cells stained for HA or CD44 shown in red. DAPI shown in blue. Scale bar = 10µm. C. Western blot of primary specimens from AML patients (with normal or high-eIF4E) and CD34 + from healthy donors showing expression of eIF4E and Ezrin. b-Actin is provided as a loading control. Each lane represents a different individual. D. Adhesion (left) and invasion (right) capacity using HS-5 stromal cells of NOMO-1 and MM6 cell lines as a function of genetic eIF4E overexpression or CRISPR knockdown respectively. Data is presented as fold change relative to their corresponding controls. Each symbol represents an independent experiment performed with replicates. The bar represents the mean with standard deviations and p-values calculated with two tailed paired T test. E. Schematic representation of colonization assay into mesenchymal stromal cell spheroid model mimicking the bone marrow niche. F. Quantification of AML cell colonization capacity relative to their corresponding controls. Each symbol represents an experimental replicate. The bar represents the mean with standard deviations and p-values, Welch’s t test.

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: Over Expression, Plasmid Preparation, Control, CRISPR, Western Blot, Staining, Expressing, Knockdown, Two Tailed Test

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

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

A. Genetic reduction of Ezrin and eIF4E using siRNA in MM6 cells grown in suspension impacts production of factors in the Ezrin-CD44-HA axis compared to the siLUC control shown in a representative western blot (left panel). b-Actin is provided as loading control. Right panel, quantification of protein expression for the indicated siRNAs relative to siLUC is shown. The expression of each protein was calculated relative to b-Actin. Each symbol represents a biological replicate. Means, standard deviations and p-values (multiple paired t tests). B. Endogenous Ezrin and eIF4E immunoprecipitations (IP) in THP-1 total cell lysates show similar observations to MM6 cells . SN supernatant after immunoprecipitation, IgG negative control. H2B also serves as a negative control for eIF4E and Ezrin IPs. C. Cytoplasmic (left) and total (right) cell lysates demonstrate Ezrin immunoprecipitated with the translation machinery including eIF4AI/II. D. Example fractionation controls for suspension (left) or invaded (right) cells shown in indicating quality of fractions. MEK and CalR are cytoplasmic markers; NOPP140 and H3K27A are nuclear markers.

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. Genetic reduction of Ezrin and eIF4E using siRNA in MM6 cells grown in suspension impacts production of factors in the Ezrin-CD44-HA axis compared to the siLUC control shown in a representative western blot (left panel). b-Actin is provided as loading control. Right panel, quantification of protein expression for the indicated siRNAs relative to siLUC is shown. The expression of each protein was calculated relative to b-Actin. Each symbol represents a biological replicate. Means, standard deviations and p-values (multiple paired t tests). B. Endogenous Ezrin and eIF4E immunoprecipitations (IP) in THP-1 total cell lysates show similar observations to MM6 cells . SN supernatant after immunoprecipitation, IgG negative control. H2B also serves as a negative control for eIF4E and Ezrin IPs. C. Cytoplasmic (left) and total (right) cell lysates demonstrate Ezrin immunoprecipitated with the translation machinery including eIF4AI/II. D. Example fractionation controls for suspension (left) or invaded (right) cells shown in indicating quality of fractions. MEK and CalR are cytoplasmic markers; NOPP140 and H3K27A are nuclear markers.

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: Suspension, Control, Western Blot, Expressing, Immunoprecipitation, Negative Control, Fractionation

A. Western blots of total cell lysates for MM6 cells in suspension treated with puromycin and analyzed by WB with anti-puromycin antibody (to assess global protein synthesis) reveal that Ezrin does not influence global translation: RNAi knockdown to Ezrin (siEZR) did not show difference compared to luciferase (siLUC) control (left panel). In contrast, CRISPR 4E reduces global translation signal by about 50% relative to CRISPR-CTRL, consistent with previous studies. Each symbol represents a biological replicate, bar showing mean, standard deviation, p-value (two tailed paired t test). B. Confocal micrographs for VISTA-R and the indicated proteins for MM6 cells upon genetic knockdowns siEIF4E), siEZR or siLUC control Arrows indicate T-PODs. Scale bar = 10µm. Western blots confirmed knockdown (Supplementary Figure 6C) C. As in B but monitoring eIF4E in T-PODs as a function of Ezrin and eIF4E knockdown. Ezrin is also reduced by eIF4E knockdown in invaded cells (Supplementary Figure 6C). Scale bar = 10µm. D. Quantitation of intensity of factors as noted in T-PODs over 3 biological replicates for VISTA-R and rRNA, 5 for Ezrin and 2 for eIF4E. Boxplots show Imaris-quantified TPOD-associated intensities of VISTA-R, rRNA, Ezrin, and eIF4E under the conditions shown. Each dot represents an individual T-POD object, p-values calculated using one way ANOVA test.

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 blots of total cell lysates for MM6 cells in suspension treated with puromycin and analyzed by WB with anti-puromycin antibody (to assess global protein synthesis) reveal that Ezrin does not influence global translation: RNAi knockdown to Ezrin (siEZR) did not show difference compared to luciferase (siLUC) control (left panel). In contrast, CRISPR 4E reduces global translation signal by about 50% relative to CRISPR-CTRL, consistent with previous studies. Each symbol represents a biological replicate, bar showing mean, standard deviation, p-value (two tailed paired t test). B. Confocal micrographs for VISTA-R and the indicated proteins for MM6 cells upon genetic knockdowns siEIF4E), siEZR or siLUC control Arrows indicate T-PODs. Scale bar = 10µm. Western blots confirmed knockdown (Supplementary Figure 6C) C. As in B but monitoring eIF4E in T-PODs as a function of Ezrin and eIF4E knockdown. Ezrin is also reduced by eIF4E knockdown in invaded cells (Supplementary Figure 6C). Scale bar = 10µm. D. Quantitation of intensity of factors as noted in T-PODs over 3 biological replicates for VISTA-R and rRNA, 5 for Ezrin and 2 for eIF4E. Boxplots show Imaris-quantified TPOD-associated intensities of VISTA-R, rRNA, Ezrin, and eIF4E under the conditions shown. Each dot represents an individual T-POD object, p-values calculated using one way ANOVA test.

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, Control, CRISPR, Standard Deviation, Two Tailed Test, Quantitation Assay

A . Schematic comparing previous strategies to monitor in situ translation based on surrogate markers of translation such as labelled amino acids (left), and our novel method for direct measurement of translation, VISTA-R (right). B. VISTA-R signals in MM6 cells are directly related to translation activity since these are abrogated upon addition of translation inhibitors. Puromycin and/or HHT were added prior to the fixation step in VISTA-R. Representative confocal micrograph of VISTA-R signal in vehicle control, puromycin, HHT or combination treated cells. DAPI is shown in blue. Scale bar = 10 µm. C. Quantitation showing means, standard deviations of the mean, data points from each measurement, p-values calculated with one way ANOVA test from over 60 cells. D. T-PODs (as shown by arrows) contain active ribosomes as seen by VISTA-R (green). VISTA-R and rRNA (red), Ezrin (blue) overlay in these T-POD. E. Same as D but demonstrating VISTA-R (green), Ezrin (magenta) and RPS6 (red) and CD44 (turquoise) are present in the same T-PODs (see arrows). F. Same as D-E but demonstrating that VISTA-R (green), eIF4E (red) and Ezrin (magenta) and CD44 (turquoise) are present in the same T-PODs (see arrows). All confocal micrographs represent a single section through the plane of the cell. Scale bars = 10 µm.

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 . Schematic comparing previous strategies to monitor in situ translation based on surrogate markers of translation such as labelled amino acids (left), and our novel method for direct measurement of translation, VISTA-R (right). B. VISTA-R signals in MM6 cells are directly related to translation activity since these are abrogated upon addition of translation inhibitors. Puromycin and/or HHT were added prior to the fixation step in VISTA-R. Representative confocal micrograph of VISTA-R signal in vehicle control, puromycin, HHT or combination treated cells. DAPI is shown in blue. Scale bar = 10 µm. C. Quantitation showing means, standard deviations of the mean, data points from each measurement, p-values calculated with one way ANOVA test from over 60 cells. D. T-PODs (as shown by arrows) contain active ribosomes as seen by VISTA-R (green). VISTA-R and rRNA (red), Ezrin (blue) overlay in these T-POD. E. Same as D but demonstrating VISTA-R (green), Ezrin (magenta) and RPS6 (red) and CD44 (turquoise) are present in the same T-PODs (see arrows). F. Same as D-E but demonstrating that VISTA-R (green), eIF4E (red) and Ezrin (magenta) and CD44 (turquoise) are present in the same T-PODs (see arrows). All confocal micrographs represent a single section through the plane of the cell. Scale bars = 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: In Situ, Activity Assay, Control, Quantitation Assay

A. Schema of VISTA-R for invaded cells. Steps for isolated invaded AML cells for marking active ribosomes with the VISTA-R method. B. Confocal microscopy for patient specimen, AML23, demonstrated active translation, characterized by VISTA-R and Ezrin are present in the same pseudopods (white arrow). A single section through the plane of the cell is shown. Scale bar = 10µm. C. Representative western blot demonstrating that protein reduction due to siRNA to eIF4E or Ezrin occurs in invaded cells, thus invaded cells are not a result of rescue from the siRNA. Results are similar to MM6 cells grown in suspension . D. Visualization of masks generated during Imaris confocal analysis to automate identification of pseudopods and subsequent measurement of contents. Scale bar = 10µm. E. Total mRNA (left) and rRNA (right) levels detected by RT-qPCR in invaded MM6 cells as a function of genetic knockdown using siRNA to EIF4E or EZR as compared to siLUC treated cells. Each symbol represents a biological replicate performed independently. Bars represent the mean, shown with standard deviations and p-values (Welch’s t test). F. Representative western blots of invaded CRISPR MM6 cells showing reduced levels of factors in the Ezrin-CD44-HA axis similar to cells grown in suspension.

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. Schema of VISTA-R for invaded cells. Steps for isolated invaded AML cells for marking active ribosomes with the VISTA-R method. B. Confocal microscopy for patient specimen, AML23, demonstrated active translation, characterized by VISTA-R and Ezrin are present in the same pseudopods (white arrow). A single section through the plane of the cell is shown. Scale bar = 10µm. C. Representative western blot demonstrating that protein reduction due to siRNA to eIF4E or Ezrin occurs in invaded cells, thus invaded cells are not a result of rescue from the siRNA. Results are similar to MM6 cells grown in suspension . D. Visualization of masks generated during Imaris confocal analysis to automate identification of pseudopods and subsequent measurement of contents. Scale bar = 10µm. E. Total mRNA (left) and rRNA (right) levels detected by RT-qPCR in invaded MM6 cells as a function of genetic knockdown using siRNA to EIF4E or EZR as compared to siLUC treated cells. Each symbol represents a biological replicate performed independently. Bars represent the mean, shown with standard deviations and p-values (Welch’s t test). F. Representative western blots of invaded CRISPR MM6 cells showing reduced levels of factors in the Ezrin-CD44-HA axis similar to cells grown in suspension.

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: Isolation, Confocal Microscopy, Western Blot, Suspension, Generated, Quantitative RT-PCR, Knockdown, CRISPR

A. Ezrin and eIF4E RIPs from cytoplasmic fractions from invaded MM6 cells compared to input. Each symbol represents a biological replicate. Means, standard deviations, p-values (Welch’s t test) are shown. Mitochondrial encoded mRNAs were used as negative controls (MT-CYB and MT-CO1). B. Representative western blots showing the expression for the indicated proteins using total cell lysates of invaded MM6 cells with knockdown of Ezrin (siEZR) or eIF4E (siEIF4E) and siLUC control. Quantitation of these western blots shown in C , each symbol represents a biological replicate, bar shows the mean, standard deviations and p-values (multiple unpaired t tests). D. Translation inhibitors decrease the adhesion capacity of MM6 cells. Each dot represents a biological replicate. Means, standard deviations and p-values (one way ANOVA) are shown.

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. Ezrin and eIF4E RIPs from cytoplasmic fractions from invaded MM6 cells compared to input. Each symbol represents a biological replicate. Means, standard deviations, p-values (Welch’s t test) are shown. Mitochondrial encoded mRNAs were used as negative controls (MT-CYB and MT-CO1). B. Representative western blots showing the expression for the indicated proteins using total cell lysates of invaded MM6 cells with knockdown of Ezrin (siEZR) or eIF4E (siEIF4E) and siLUC control. Quantitation of these western blots shown in C , each symbol represents a biological replicate, bar shows the mean, standard deviations and p-values (multiple unpaired t tests). D. Translation inhibitors decrease the adhesion capacity of MM6 cells. Each dot represents a biological replicate. Means, standard deviations and p-values (one way ANOVA) are shown.

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, Expressing, Knockdown, Control, Quantitation Assay

a) Ezrin protein sequence: membrane binding FERM domain consists of three lobes – F1 (residues P2-P86), F2 (residues E87-G202), and F3 (residues I203-K296); linker region consists of residues P297-P496; and the actin-binding C-terminal domain (CTD) consists of residues T497-L586. b) AlphaFold 3.0 predicted closed-form structure of the full-length ezrin protein . In the predicted closed-form ezrin structure, C-terminal CTD domain interacts with FERM over the F2-F3 subdomain surface. c) System setup for the FERM-CTD structure (PDB ID: 4RM9) at a DOPC:DOPS:PIP 2 (80:16:4ratio) membrane that was used in the molecular dynamics simulations in this study. d) Number of PIP 2 head groups phosphorus atoms within 10 Å of any atoms in the residues in FERM. Asterisks (*) indicate significant difference (α = 0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns, e) Aggregation of PIP 2 (orange), DOPS (blue) and DOPC (gray) phospholipids in a PIP 2 /DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark. f) Number of DOPS within 10 Å of the residues in FERM. Asterisks (*) indicate significant difference (independent sample t-test, α=0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns. Double asterisk (**) indicates significant difference (ANOVA, α = 0.05) between the means in systems with DOPC/DOPS/PIP 2 membranes and DOPC/DOPS membranes in the 990-1000 ns interval. g) Aggregation of DOPS phospholipids (blue) in a DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark.

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Ezrin protein sequence: membrane binding FERM domain consists of three lobes – F1 (residues P2-P86), F2 (residues E87-G202), and F3 (residues I203-K296); linker region consists of residues P297-P496; and the actin-binding C-terminal domain (CTD) consists of residues T497-L586. b) AlphaFold 3.0 predicted closed-form structure of the full-length ezrin protein . In the predicted closed-form ezrin structure, C-terminal CTD domain interacts with FERM over the F2-F3 subdomain surface. c) System setup for the FERM-CTD structure (PDB ID: 4RM9) at a DOPC:DOPS:PIP 2 (80:16:4ratio) membrane that was used in the molecular dynamics simulations in this study. d) Number of PIP 2 head groups phosphorus atoms within 10 Å of any atoms in the residues in FERM. Asterisks (*) indicate significant difference (α = 0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns, e) Aggregation of PIP 2 (orange), DOPS (blue) and DOPC (gray) phospholipids in a PIP 2 /DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark. f) Number of DOPS within 10 Å of the residues in FERM. Asterisks (*) indicate significant difference (independent sample t-test, α=0.05) between the means of contact counts between 0-10 ns and 495-505 ns or 990-1000 ns. Double asterisk (**) indicates significant difference (ANOVA, α = 0.05) between the means in systems with DOPC/DOPS/PIP 2 membranes and DOPC/DOPS membranes in the 990-1000 ns interval. g) Aggregation of DOPS phospholipids (blue) in a DOPS/DOPC membrane at the F1-F3 surface of ezrin FERM domain at t = 1000 ns mark.

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Sequencing, Membrane, Binding Assay

a) Hydrogen bond counts between ezrin F2-CTD lobes (left) and ezrin F3-CTD lobes (right). Significantly more hydrogen bonds are broken in the system where ezrin has a phosphorylated T567 residue. b) Significant opening between CTD residues AA538-AA546 and FERM F2 lobe is only observed in the FERM-CTD system with phosphorylated T567 at a PIP 2 /DOPS/DOPC membrane. Left – crystal structure of ezrin FERM and C-ERMAD (PDB ID:4RM9). Right – distance between alpha carbon atoms of residue pairs K162-R542, Q160-K546, R156-T548 in the beginning of the equilibrated unbiased MD simulations (t=0 ns) and the end of the 1 µs simulation (t=1000 ns) for versions of the system that contain either a nonphosphorylated T567 (npT567, top) or phosphorylated T567 (pT567, bottom). b)-d) Distances of CTD residue Cα atoms from the Cα atoms of their nearest FERM F2-F3 residues. b) distances of CTD residues AA538-AA542 increase only for a system phosphorylated T567 at a PIP 2 /DOPS/DOPC membrane (red), c) large changes in distances are observed between Cα atoms of CTD helix H3 (residues AA549-AA559) and FERM F3 residues K230-T235 an CTD helix H3, d) no significant change in distance is observed between CTD helix H5 (residues T576-L586) and nearest FERM atoms. e) Coulombic potentials between CTD helices H1-H5 and nearest secondary structures in FERM F2-F3 lobes. Helices H1-H5 correspond to amino acid residues as follows: H1 – residues E525-Q540, H2 – residues A541-R547, H3 – residues H549-R559, H4 – residues K564-R572, H5 – residues T576-L586. Notation: pT567 – system with a phosphorylated T567, wt – system with a nonphosphorylated T567, soln – simulation carried out in a KCl solution. Asterisks (*) indicate a statistically significant coulombic attraction strength difference for a certain CTD helix between the FERM-CTD pT567 system at a PIP 2 /DOPC/DOPS membrane against any other systems. The inset provides a visualization of H1-H5 CTD helices in the crystal structure of closed state ezrin (PDB ID: 4RM9).

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Hydrogen bond counts between ezrin F2-CTD lobes (left) and ezrin F3-CTD lobes (right). Significantly more hydrogen bonds are broken in the system where ezrin has a phosphorylated T567 residue. b) Significant opening between CTD residues AA538-AA546 and FERM F2 lobe is only observed in the FERM-CTD system with phosphorylated T567 at a PIP 2 /DOPS/DOPC membrane. Left – crystal structure of ezrin FERM and C-ERMAD (PDB ID:4RM9). Right – distance between alpha carbon atoms of residue pairs K162-R542, Q160-K546, R156-T548 in the beginning of the equilibrated unbiased MD simulations (t=0 ns) and the end of the 1 µs simulation (t=1000 ns) for versions of the system that contain either a nonphosphorylated T567 (npT567, top) or phosphorylated T567 (pT567, bottom). b)-d) Distances of CTD residue Cα atoms from the Cα atoms of their nearest FERM F2-F3 residues. b) distances of CTD residues AA538-AA542 increase only for a system phosphorylated T567 at a PIP 2 /DOPS/DOPC membrane (red), c) large changes in distances are observed between Cα atoms of CTD helix H3 (residues AA549-AA559) and FERM F3 residues K230-T235 an CTD helix H3, d) no significant change in distance is observed between CTD helix H5 (residues T576-L586) and nearest FERM atoms. e) Coulombic potentials between CTD helices H1-H5 and nearest secondary structures in FERM F2-F3 lobes. Helices H1-H5 correspond to amino acid residues as follows: H1 – residues E525-Q540, H2 – residues A541-R547, H3 – residues H549-R559, H4 – residues K564-R572, H5 – residues T576-L586. Notation: pT567 – system with a phosphorylated T567, wt – system with a nonphosphorylated T567, soln – simulation carried out in a KCl solution. Asterisks (*) indicate a statistically significant coulombic attraction strength difference for a certain CTD helix between the FERM-CTD pT567 system at a PIP 2 /DOPC/DOPS membrane against any other systems. The inset provides a visualization of H1-H5 CTD helices in the crystal structure of closed state ezrin (PDB ID: 4RM9).

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Residue, Membrane

a) Well-tempered metadynamics contact map collective variable can be successfully used to make the ezrin system undergo a full dissociation between FERM and CTD domains in about 20 ns. Image on the right is the schematic of how the contact map CV includes contacts across the entire surface of CTD and FERM F2-F3 lobes. b) The free energy surfaces of well-tempered contact map WTMetaD show that ezrin system with nonphosphorylated T567 has a mean barrier of 11.2 ± 0.3 kcal/mol for the transition from closed to dissociated FERM-CTD (blue) and a 3.2 ± 0.5 kcal/mol transition barrier between the closed and open states when the system has a phosphorylated T567 residue. The barrier of EBP50-FERM dissociation (11.7 ± 1.1 kcal/mol) is similar to that of FERM-wtCTD. Results are reported as mean (± SE) of sample free energy values corresponding to certain contact map collective variable values. The free energy profiles converged in 50-100 ns of the WTMetaD runs (Supplementary Figure S8). c) Ezrin FERM-CTD crystal structure (PDB ID: 4RM9). d) Crystal structure of ezrin FERM domain and EBP50 C-terminal residues (PDB ID: 1SGH). The C-terminal end of ezrin CTD domain has very similar alignment to F3 lobe of FERM as does the end helix of EBP50. e)-f) Distance between the C α atoms of different contact map pairs at contact map values 0.1-0.9 obtained from WTMetaD runs between F3 and CTD (e), and between F2 and CTD (f). Results in e)-f) are presented as mean distance and standard error for each of the contact map coordinate values collected from all WTMetaD simulation frames that have a contact map value in a certain range. g) RMSF plots for lobe F2 (left) and F3 (right) during the WTMetaD simulations for the system with phosphorylated T567 (red) and the system with nonphosphorylated T567 (blue).

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Well-tempered metadynamics contact map collective variable can be successfully used to make the ezrin system undergo a full dissociation between FERM and CTD domains in about 20 ns. Image on the right is the schematic of how the contact map CV includes contacts across the entire surface of CTD and FERM F2-F3 lobes. b) The free energy surfaces of well-tempered contact map WTMetaD show that ezrin system with nonphosphorylated T567 has a mean barrier of 11.2 ± 0.3 kcal/mol for the transition from closed to dissociated FERM-CTD (blue) and a 3.2 ± 0.5 kcal/mol transition barrier between the closed and open states when the system has a phosphorylated T567 residue. The barrier of EBP50-FERM dissociation (11.7 ± 1.1 kcal/mol) is similar to that of FERM-wtCTD. Results are reported as mean (± SE) of sample free energy values corresponding to certain contact map collective variable values. The free energy profiles converged in 50-100 ns of the WTMetaD runs (Supplementary Figure S8). c) Ezrin FERM-CTD crystal structure (PDB ID: 4RM9). d) Crystal structure of ezrin FERM domain and EBP50 C-terminal residues (PDB ID: 1SGH). The C-terminal end of ezrin CTD domain has very similar alignment to F3 lobe of FERM as does the end helix of EBP50. e)-f) Distance between the C α atoms of different contact map pairs at contact map values 0.1-0.9 obtained from WTMetaD runs between F3 and CTD (e), and between F2 and CTD (f). Results in e)-f) are presented as mean distance and standard error for each of the contact map coordinate values collected from all WTMetaD simulation frames that have a contact map value in a certain range. g) RMSF plots for lobe F2 (left) and F3 (right) during the WTMetaD simulations for the system with phosphorylated T567 (red) and the system with nonphosphorylated T567 (blue).

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Residue

a) Coomassie staining of purified proteins isolated from either HEK-293T cells (LOK-GFP-Flag) or Bacterial expression (EBP50 constructs and Ezrin). b) Western blotting of biochemical in vitro kinase assays blotting for total Ezrin vs. Ezrin phosphorylated at T567 (pT567). Purified full length untagged Ezrin and LOK-GFP-Flag kinase and were added at a constant concentration 1.5mg/ml and 0.05mg/ml respectively. Prior to the addition of ATP, the proteins were incubated with 0.5mg/ml of EBP50 or EBP50 variants and PI(4,5)P 2 was either added or withheld from duplicates of each EBP50 condition. Mutations in the two PDZ domains activate EBP50 leading to constitutively active binding between EBP50 and Ezrin’s FERM domain while EBP50’s tail domain is an unregulated Ezrin FERM binding motif. Phosphorylation of Ezrin at pT567 was PI(4,5)P 2 dependent but independent from EBP50 binding. c) Western blotting of lysate from human WT Jeg-3 epithelial cells and Jeg-3 CRISPR knockouts of EBP50 and LOK/SLK. Cells were treated with either the phosphatase inhibitor Calyculin A (Cal. A), the kinase inhibitor Staurosporine (Staursp) or no treatment prior to lysing and blotted for total Ezrin vs. Ezrin phosphorylated at T567 (pT567). Cells lacking EBP50 have less pT567 than their WT counterparts. The effect of treatment with Cal. A or Staursp. is not altered in cells lacking EBP50 vs. WT cells indicating that EBP50 influences the turnover but not the capacity of phosphorylation/dephosphorylation of ezrin at T567. Cells lacking the endogenous ERM kinases LOK/SLK have no phosphorylation at T567.

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a) Coomassie staining of purified proteins isolated from either HEK-293T cells (LOK-GFP-Flag) or Bacterial expression (EBP50 constructs and Ezrin). b) Western blotting of biochemical in vitro kinase assays blotting for total Ezrin vs. Ezrin phosphorylated at T567 (pT567). Purified full length untagged Ezrin and LOK-GFP-Flag kinase and were added at a constant concentration 1.5mg/ml and 0.05mg/ml respectively. Prior to the addition of ATP, the proteins were incubated with 0.5mg/ml of EBP50 or EBP50 variants and PI(4,5)P 2 was either added or withheld from duplicates of each EBP50 condition. Mutations in the two PDZ domains activate EBP50 leading to constitutively active binding between EBP50 and Ezrin’s FERM domain while EBP50’s tail domain is an unregulated Ezrin FERM binding motif. Phosphorylation of Ezrin at pT567 was PI(4,5)P 2 dependent but independent from EBP50 binding. c) Western blotting of lysate from human WT Jeg-3 epithelial cells and Jeg-3 CRISPR knockouts of EBP50 and LOK/SLK. Cells were treated with either the phosphatase inhibitor Calyculin A (Cal. A), the kinase inhibitor Staurosporine (Staursp) or no treatment prior to lysing and blotted for total Ezrin vs. Ezrin phosphorylated at T567 (pT567). Cells lacking EBP50 have less pT567 than their WT counterparts. The effect of treatment with Cal. A or Staursp. is not altered in cells lacking EBP50 vs. WT cells indicating that EBP50 influences the turnover but not the capacity of phosphorylation/dephosphorylation of ezrin at T567. Cells lacking the endogenous ERM kinases LOK/SLK have no phosphorylation at T567.

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Staining, Purification, Isolation, Expressing, Construct, Western Blot, In Vitro, Concentration Assay, Incubation, Binding Assay, Phospho-proteomics, CRISPR, De-Phosphorylation Assay

a)-b) Closed state ezrin is attracted to apical cell membranes due to highly negatively charged PIP 2 phospholipids. c) Upon attachment to membrane, FERM F1 and F3 subdomains recruit more PIP 2 . d) Ezrin wtCTD spontaneously dissociates from FERM over long timescales. e) Dissociation of CTD leaves space between FERM and CTD for LOK kinase to phosphorylate the T567 residue. f) Upon T567 phosphorylation, ezrin primarily remains in the open state due to the lower propensity to inhabit the closed state. g) CTD is enabled to float away and interact with actin filaments, thus leaving space for downstream FERM F2-F3 interactions with EBP50 or other cellular targets.

Journal: bioRxiv

Article Title: On the Mechanism of Ezrin Activation

doi: 10.1101/2025.11.07.687285

Figure Lengend Snippet: a)-b) Closed state ezrin is attracted to apical cell membranes due to highly negatively charged PIP 2 phospholipids. c) Upon attachment to membrane, FERM F1 and F3 subdomains recruit more PIP 2 . d) Ezrin wtCTD spontaneously dissociates from FERM over long timescales. e) Dissociation of CTD leaves space between FERM and CTD for LOK kinase to phosphorylate the T567 residue. f) Upon T567 phosphorylation, ezrin primarily remains in the open state due to the lower propensity to inhabit the closed state. g) CTD is enabled to float away and interact with actin filaments, thus leaving space for downstream FERM F2-F3 interactions with EBP50 or other cellular targets.

Article Snippet: Ezrin mouse monoclonal antibody (DSHB cat# CPTC-Ezrin-1) was used at a dilution of 1:5000 and Phospho-ezrin was detected using rabbit anti-pT567 antibody, raised against recombinant phosphopeptide CRDKYK(pT)LRQIR ( ) was used at a dilution of 1:1000.

Techniques: Membrane, Residue, Phospho-proteomics