vimentin apex2 in pecfp  (Addgene inc)

Journal: Journal of Proteome Research

Article Title: Proximity Labeling Reveals How Lrp2 Interacts with the Endocytic Machine

doi: 10.1021/acs.jproteome.5c01053

Figure Lengend Snippet: NGAL protein immunoblot shows that urine NGALa biomarker of kidney injuryis not elevated in Lrp2 APEX2‑‑V5/+ mice. Comparison with the deletion of Lrp2 in the proximal tubule of the kidney. Equal amounts of urine creatinine were loaded onto each lane. F, female M, male.

Article Snippet: The LRP2 APEX2‐V5 mouse has been deposited at Jackson Laboratories as JAX Strain name: Lrp2-APEX2; Jax Strain ID: 419746.

Techniques: Western Blot, Biomarker Discovery, Comparison

Journal: Journal of Proteome Research

Article Title: Proximity Labeling Reveals How Lrp2 Interacts with the Endocytic Machine

doi: 10.1021/acs.jproteome.5c01053

Figure Lengend Snippet: Proximal tubules are biotinylated in vitro and in vivo. (A) Lrp2 APEX‑V52/+ proximal tubule membrane fractions were biotinylated in vitro in the presence of H 2 O 2 , and biotinylated proteins were precipitated with streptavidin magnetic beads. The samples were analyzed by Western blot using HRP-conjugated streptavidin (1:5000). (B) Renal artery was cannulated and perfused with bromophenol blue (1 mg/mL), producing blue urine filling the bladder. (C) Lrp2 APEX2‑V5/+ kidney was perfused with biotin phenol and H 2 O 2 , and biotinylated proteins were precipitated with streptavidin magnetic beads. The samples were analyzed by Western blotting using HRP-conjugated streptavidin (1:5000). (D) Expression of the LRP2 APEX2‑V5 fusion protein in vivo. Note that Lrp2 and V5 immunoreactivity colocalize with biotinylation, which is detected by apical staining with 488-Avidin in the proximal tubule.

Article Snippet: The LRP2 APEX2‐V5 mouse has been deposited at Jackson Laboratories as JAX Strain name: Lrp2-APEX2; Jax Strain ID: 419746.

Techniques: In Vitro, In Vivo, Membrane, Magnetic Beads, Western Blot, Expressing, Staining, Avidin-Biotin Assay

Journal: Journal of Proteome Research

Article Title: Proximity Labeling Reveals How Lrp2 Interacts with the Endocytic Machine

doi: 10.1021/acs.jproteome.5c01053

Figure Lengend Snippet: Specificity of LRP2 APEX2‑V5/+ . Fresh, unfixed kidney thick sections (0.5 mm) were treated with biotin-phenol. (A) Wild-type kidney sections. Note the absence of both the V5 c-terminal tag and biotinylation (streptavidin staining). (B) Lrp2 APEX2‑V5/+ kidney sections depict both the V5 c-terminal tag and biotinylation (streptavidin staining). Biotinylation exclusively colocalizes with Lrp2 and V5 immunoreactivity in the apical domain of the proximal tubule. Biotinylation is dependent on H 2 O 2 .

Article Snippet: The LRP2 APEX2‐V5 mouse has been deposited at Jackson Laboratories as JAX Strain name: Lrp2-APEX2; Jax Strain ID: 419746.

Techniques: Staining

Journal: Journal of Proteome Research

Article Title: Proximity Labeling Reveals How Lrp2 Interacts with the Endocytic Machine

doi: 10.1021/acs.jproteome.5c01053

Figure Lengend Snippet: Localization of LRP2 APEX2‑V5/+ biotinylated proteins. Lrp2 C-terminal-dependent biotinylation in vivo identified cytoplasmic neighbors. Colocalization of biotinylation (detected by 488-avidin) with target proteins and with the V5 tag begins at the tubular pole of the glomerulus at the initiation of the proximal tubule epithelia (top panel). Colocalized protein refers to staining for the proteins named on the left side of the lower panels.

Article Snippet: The LRP2 APEX2‐V5 mouse has been deposited at Jackson Laboratories as JAX Strain name: Lrp2-APEX2; Jax Strain ID: 419746.

Techniques: In Vivo, Avidin-Biotin Assay, Staining

Journal: Journal of Proteome Research

Article Title: Proximity Labeling Reveals How Lrp2 Interacts with the Endocytic Machine

doi: 10.1021/acs.jproteome.5c01053

Figure Lengend Snippet: Immuno-isolation of endosomes containing c-terminal Lrp2 APEX‑V5 . (A) Kidney membrane fractions were prepared on an OptiPrep step gradient (top two steps are shown). (B) Bradford protein assay of each gradient fraction. (C) Lrp2 immunoblot of gradient fractions (with equal protein loading). Lrp2 is concentrated in lanes 6–8. (D) Western blot showing immuno-isolation of Lrp2 APEX‑V5 endosomes from OptiPrep fractions 6–8 using anti-V5 nanobody beads: Lane 1 = OptiPrep fractions; Lane 2 = nanobody bead flow-through; Lane 3 = Capture of V5 + , Lrp2 + , Cubn + , Alb + endosomes. (E) Western blot showing introduction of myoglobin prior to kidney fractionation on OptiPrep step gradients and anti-V5 nanobody bead pulldowns from different pooled OptiPrep fractions (2−3, 4−5, 6−7, 8−9, 10−11). Myoglobin is found in Lrp2 + endosomes along with the Lrp2 endocytic adaptor protein, DAB2.

Article Snippet: The LRP2 APEX2‐V5 mouse has been deposited at Jackson Laboratories as JAX Strain name: Lrp2-APEX2; Jax Strain ID: 419746.

Techniques: Isolation, Membrane, Bradford Protein Assay, Western Blot, Fractionation

Journal: Frontiers in Oncology

Article Title: A novel anti-CD20, concabody, enhances immunotherapy efficacy by targeting MPZL1 and augmenting antibody-induced cell death

doi: 10.3389/fonc.2026.1748576

Figure Lengend Snippet: Workflow of APEX2-mediated labeling to identify proteins near obinutuzumab-bound CD20. (A) Schematic overview of the APEX2-based proximity-labeling system and experimental workflow to identify proteins in the vicinity of the anti-CD20 mAb–CD20 complex. (B) Design of APEX2-fused recombinant antibodies and validation of the purified antibodies by SDS-PAGE. These recombinant APEX2-fused antibodies carry a 4×G4S linker (~2kDa) between the Fc region and APEX2 (2nd generation APEX2 design). R, rituximab; C-A, isotype control (OBI-LC/TRA-HC)-APEX2; R-A, rituximab-APEX2; O-A, obinutuzumab-APEX2. (C) CD20 binding ability of wild-type and APEX2-fused anti-CD20 mAbs in Raji cells (n=3). (D) Antibody-induced direct cell death (DCD) activity of the wild-type and APEX2-fused antibodies in Raji cells (n=3). (E) Western blot analysis of biotinylated proteins using APEX2-fused antibodies was performed in Raji cells. Representative blots are shown (n=2). (F) Silver staining of biotinylated proteins. Representative blots are shown (n=2). (G) Pipeline for candidate protein selection from proximity-labeling mass spectrometry data. (H) Subcellular localization of identified candidate Proteins. Localization was annotated using the GeneCards and NCBI databases. (I) Subcellular localization of proteins predominantly enriched by rituximab and obinutuzumab. All data are presented as the mean ± SD. Statistical analysis was performed using the Welch’s t-test. Significance is indicated as follows: ns, not significant.

Article Snippet: The cells were then incubated with the indicated concentrations of anti-CD20 antibodies (OBI, RTX), anti-CD20-APEX2 fusion antibodies (O-A, R-A), and control antibodies (C-A) at 4 °C for 30 min. After washing the cells with PBS, the cell pellets were resuspended in 50 μL of PBS containing an anti-human Ig Fc-specific FITC-conjugated secondary antibody at a 1:500 dilution (Jackson Laboratories; Cat No.109-095-008).

Techniques: Labeling, Recombinant, Biomarker Discovery, Purification, SDS Page, Control, Binding Assay, Activity Assay, Western Blot, Silver Staining, Selection, Mass Spectrometry

Journal: Frontiers in Oncology

Article Title: A novel anti-CD20, concabody, enhances immunotherapy efficacy by targeting MPZL1 and augmenting antibody-induced cell death

doi: 10.3389/fonc.2026.1748576

Figure Lengend Snippet: MPZL1 is a novel therapeutic target identified through its role in OBI-induced lysosomal membrane permeabilization and direct cell death. (A) Validation of doxycycline-inducible gene knockdown in Raji cells using qPCR (n=3). shRNAs targeting the indicated genes were induced with doxycycline; shLUC (luciferase) and shCD20 were used as negative and positive controls, respectively. (B) Quantification of lysosomal membrane permeabilization (LMP) following OBI treatment in candidate knockdown cells (n=4). LMP was measured as the proportion of cells exhibiting reduced LysoTracker Green fluorescence. DOX+, doxycycline-induced knockdown; DOX−, control. (C) Measurement of OBI-induced direct cell death (DCD) in candidate knockdown cells showing altered LMP, compared to shLUC controls (n=3). DCD was assessed by propidium iodide (PI) staining. (D) Time-course analysis of OBI-induced DCD (21 nM) in MPZL1 knockdown cells (sh3 from panel C) following doxycycline induction(n=3). (E) Analysis of CD20 expression by RTX binding in the MPZL1 knockdown cell line(n=3). (F) Western blot validation of MPZL1 knockdown (DOX+) and overexpression (MZPL1 OX) by CRISPRa. EV, empty vector. Band intensities were quantified using ImageJ. Representative blots are shown (n=2). (G) qPCR analysis of MPZL1 expression in MPZL1-overexpressing cell lines (n=3). (H) Enhanced OBI-induced DCD in MPZL1-overexpressing cell lines (n=3). (I) Dose-dependent LMP induced by concanavalin A (Con A) in Raji cells. (J–L) Reduced Con A-induced LMP (J) , DCD (K) , and Con A-internalization (L) in MPZL1 knockdown cell lines (n=3). Con A (100nM) was used in all three panels. Data are presented as mean ± SD. Statistical analysis was performed using Welch’s t-test. Significance is indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The cells were then incubated with the indicated concentrations of anti-CD20 antibodies (OBI, RTX), anti-CD20-APEX2 fusion antibodies (O-A, R-A), and control antibodies (C-A) at 4 °C for 30 min. After washing the cells with PBS, the cell pellets were resuspended in 50 μL of PBS containing an anti-human Ig Fc-specific FITC-conjugated secondary antibody at a 1:500 dilution (Jackson Laboratories; Cat No.109-095-008).

Techniques: Membrane, Biomarker Discovery, Knockdown, Luciferase, Fluorescence, Control, Staining, Expressing, Binding Assay, Western Blot, Over Expression, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: A novel anti-CD20, concabody, enhances immunotherapy efficacy by targeting MPZL1 and augmenting antibody-induced cell death

doi: 10.3389/fonc.2026.1748576

Figure Lengend Snippet: Concabodies, anti-CD20 antibodies fused with the MPZL1-binding lectin Con A, enhance direct cell death and antibody-dependent cellular cytotoxicity. (A) Schematic design of concabody and SDS-PAGE analysis of purified concabodies. These recombinant Con A–fused antibodies carry a 4×G4S linker(~2kDa) between the Fc region and Con A (~28 kDa). O, obinutuzumab; T-C, isotype control (trastuzumab)-Con A; R-C, rituximab-Con A; O-C, obinutuzumab-Con A. (B) Dose-dependent induction of direct cell death (DCD) by wild-type antibodies (TRA, RTX, OBI) and concabodies (T-C, R-C, O-C) in Raji cells (n=3). (C) Cell death induced by TRA-Con A or free Con A in Raji cells(n=3). (D) DCD induced by OBI and OBI-Con A in CD20 knockout Raji cells. (E) Comparison of DCD induced by wild-type anti-CD20 antibodies alone, free Con A plus antibody co-treatment, and concabodies in Raji cells (n=3). (F) MPZL1 dependency of R-C– and O-C–induced DCD assessed in doxycycline-inducible MPZL1 knockdown cells (DOX− vs . DOX+) in Raji cells(n=3). (G) Internalization efficiency of concabodies compared to wild-type antibodies in Raji cells (n=4). (H) DCD induced by concabodies in MPZL1 Y241F mutant cells, in which phosphorylation-dependent signaling is disrupted in Raji cells (n=3). EV, empty vector. (I) Lysosomal membrane permeabilization(LMP) (left) and direct cell death(DCD) (right) assays were conducted in Raji cells using wild-type antibodies (T, TRA; R, RTX; O, OBI) and concabodies (T-C, TRA-Con A; R-C, RTX-Con A; O-C, OBI-Con A)(n=3). (J) Lysosomal membrane permeabilization(LMP) (left) and Direct cell death(DCD) (right) assays were conducted in Ramos cells using wild-type antibodies (T, TRA; R, RTX; O, OBI) and concabodies (T-C, TRA-Con A; R-C, RTX-Con A; O-C, OBI-Con A)(n=3). (K) Increased antibody-dependent cellular cytotoxicity (ADCC) by concabodies (T-C, R-C, O-C) compared to wild-type antibodies(n=3). ADCC was measured by co-incubating 1×10 4 Raji cells with 5×10 4 healthy donor PBMCs. (L) Enhanced B-cell depletion by concabodies compared to wild-type antibodies in ex vivo assays using PBMCs from patients with chronic lymphocytic leukemia (CLL). Data are presented as mean ± SD. Statistical significance was determined using Welch’s t-test for comparisons between parental controls and specific groups (G, H) .For comparisons between multiple conjugate groups, One-way ANOVA followed by Tukey’s post-hoc test was employed. (F–J) Significance is indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

Article Snippet: The cells were then incubated with the indicated concentrations of anti-CD20 antibodies (OBI, RTX), anti-CD20-APEX2 fusion antibodies (O-A, R-A), and control antibodies (C-A) at 4 °C for 30 min. After washing the cells with PBS, the cell pellets were resuspended in 50 μL of PBS containing an anti-human Ig Fc-specific FITC-conjugated secondary antibody at a 1:500 dilution (Jackson Laboratories; Cat No.109-095-008).

Techniques: Binding Assay, SDS Page, Purification, Recombinant, Control, Knock-Out, Comparison, Knockdown, Mutagenesis, Phospho-proteomics, Plasmid Preparation, Membrane, Ex Vivo