Journal: bioRxiv

Article Title: Leishmania exploits the macrophage endoplasmic reticulum-shaping protein CLIMP-63 to modulate mitochondrial biogenesis and bioenergetics

doi: 10.64898/2026.03.19.712868

Figure Lengend Snippet: BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

Article Snippet: The mouse anti-CLIMP-63 monoclonal antibody (sc-393544) was from Santa Cruz Biotechnology, the rabbit anti-RTN4 polyclonal antibody (ab186735) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit polyclonal antibody RTN4 (10950-1-AP) was from Proteintech, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody ( ) was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, the rabbit anti-Tom20 polyclonal antibody (EPR15581-54) was from Abcam, and the rat anti-LAMP-1 monoclonal antibody 1D4B developed by J.T.

Techniques: Control, Infection, Comparison

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Rtn4 knockdown decreases cell surface localization of integrin β1 and HLA-A. HeLa cells were transiently cotransfected with siRNA against Rtn4 and Block-iT fluorescent control or with Block-iT alone. (A) Cells were immunostained for Rtn4. (B) Rtn4 immunofluorescence intensity was quantified for 30–36 cells per condition. (C) Nonpermeabilized cells were stained for surface-localized integrin β1. (D) Nonpermeabilized cells were stained for surface-localized HLA-A. (E) Integrin β1 surface fluorescence staining intensity was quantified for 39–51 transfected and nontransfected cells per condition. (F) HLA-A surface fluorescence staining intensity was quantified for 34–51 transfected and nontransfected cells per condition. All representative images are maximum intensity projections of confocal z-stacks. Scale bars are 10 µm. Error bars represent SD. ****, p ≤ 0.0001; **, p ≤ 0.01; NS, not significant.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques: Knockdown, Blocking Assay, Control, Immunofluorescence, Staining, Fluorescence, Transfection

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Rtn4a levels modulate the kinetics of VSVG-mCherry trafficking to the cell surface. HeLa cells were transiently cotransfected with a plasmid expressing GFP-NLS as a control, a plasmid expressing Rtn4a-GFP, or siRNA against Rtn4 along with the RUSH construct Str-Ii_VSVG-SBP-mCherry. ER-trapped VSVG was released by addition of 40 µM D-Biotin to the growth media. Cells were fixed before biotin addition and at 15, 30, or 60 min after biotin addition. (A) Representative images of maximum intensity projections of confocal z-stacks showing total VSVG-mCherry. (B) Representative images of single confocal z-planes at the cell periphery to visualize cell surface–localized VSVG-mCherry. (C) Fluorescence intensity of VSVG-mCherry was quantified from the cell surface z-plane and normalized to the total VSVG-mCherry fluorescence intensity measured from the maximum intensity projection of the same cell for 8–12 cells per condition and time point. Scale bars are 10 µm. Error bars represent SD. ****, p ≤ 0.0001; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques: Plasmid Preparation, Expressing, Control, Construct, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Rtn4a levels modulate cell adhesion. HeLa cells were transiently transfected with a plasmid expressing GFP-NLS as a control, a plasmid expressing Rtn4a-GFP, or siRNA against Rtn4. Cells were allowed to attach to collagen-coated dishes for 30 min. Unattached cells were removed, and attached cells were fixed, stained with crystal violet, and lysed. Cell lysate absorbance was measured at 540 nm (see Materials and Methods ). Averages from nine independent experiments are shown. Error bars represent SD. **, p ≤ 0.01; *, p ≤ 0.05.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Staining

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques:

Journal: Cell Death and Differentiation

Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

doi: 10.1038/cdd.2011.206

Figure Lengend Snippet: Nogo-A expression in cortical neurons. (a) The diagram for antibodies against different antigen recognition sites of human Nogo-A NiR-Δ2 (amino acids (aa) 57–185), NiG-Δ20 (aa 564–749) and Nogo-66 (aa 1055–1120) are the three inhibitory regions of human Nogo-A. TMs are transmembrane domains located on the two sides of Nogo-66. The arrows indicate the target regions of five Nogo-A-specific antibodies, polyclonal antibody (pAb) A201 against aa 201–250, monoclonal antibody (mAb) A563 against aa 563–627, mAb 1E8 against aa 570–691 and pAb A620 against aa 620–1004. 2B3 is targeted to Nogo-66, aa 1055–1120. (b) Top: the expression of neuronal Nogo-A was immunostained with A620 and TuJ1, an immature neuronal marker. Lower: subcellular distribution of neuronal Nogo-A was blotted with A620. Cytosolic (F1), organelle/membrane (F2), nucleic (F3) and cytoskeleton fractions (F4) were the four subcellular fractions of neurons. (c) Living cell staining for neurons were performed with pAb A620, mAb 1E8 or pAb A201 (top). The A620 and 1E8 antibodies had previously been shown to label the extracellular amino-Nogo-A in oligodendrocytes (lower). (d) The total RNA was extracted from neurons exposed to 50 μM hydrogen peroxide (H2O2) for 8 h or not and submitted to quantitative real-time polymerase chain reaction (PCR). β-Actin was selected as an inner standard. (e) Cell lysates from neurons exposed to 50 μM H2O2 for 8 h or not were blotted with pAb A620. β-Actin was selected as an inner standard (left). Quantification by densitometric scans was presented by Nogo-A/β-actin (right). Bar=50 μm. n=3, mean±S.D., paired t-test, *P<0.05, ***P<0.001

Article Snippet: 7 , 15 For western blots, A620 and two commercial antibodies, A201 (Prosci, San Diego, CA, USA; no. 4089) and A563 (BD Biosciences PharMingen, San Diego, CA, USA; no. 612238) were used.

Techniques: Expressing, Marker, Membrane, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

doi: 10.1038/cdd.2011.206

Figure Lengend Snippet: In all, 290–562 residues are the pivotal domain of amino-Nogo-A for resisting to oxidative damage. (a) Schematic diagram of different deletions (M1–M9) of amino-Nogo. (b) Neurons were pretreated with 0.2 μM HIV-1 trans-activating (TAT)-M1-M9 for 2 h, followed by exposure to 50 μM hydrogen peroxide (H2O2) for another 12 h in the presence of TAT-M1-M9. Cell death rate was calculated by propidium iodide (PI) (+)/Hoechst (+). (c) Lysates from neurons treated by H2O2 with or without TAT-AM was performed to western blot with anti-HA and A201 antibodies. β-Actin was selected as a loading control. *Presented the nonspecific bands of anti-HA antibody. (d) HEK293FT transfected with mock or full-length human Nogo-A were treated with indicated concentrations of H2O2 for 1 h, and cell lysates were subjected to western blot probed with A201 antibody. β-Actin was selected as a loading control. The 48 kDa bands were pointed with arrow; n=4, mean±S.D., one-way analysis of variance (ANOVA), *P<0.05; ***P<0.001

Article Snippet: 7 , 15 For western blots, A620 and two commercial antibodies, A201 (Prosci, San Diego, CA, USA; no. 4089) and A563 (BD Biosciences PharMingen, San Diego, CA, USA; no. 612238) were used.

Techniques: Western Blot, Control, Transfection