rabbit polyclonal antibody rtn4  (Proteintech)

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: 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: eLife

Article Title: Rab10 regulates neuropeptide release by maintaining Ca 2+ homeostasis and protein synthesis

doi: 10.7554/eLife.94930

Figure Lengend Snippet: ( A ) Example images of control or Rab10 KD hippocampal neurons (days in vitro [DIV]14) stained for the dendrite marker MAP2 (green), two ER markers KDEL (red) and RTN4 (magenta). Scale bar: 50 μm. ( B ) Quantification of RTN4 intensity in MAP2-positive dendrites. ( C ) Quantification of KDEL intensity in MAP2-positive dendrites. ( D ) The ratio of neuritic to somatic RTN4 intensity (N/S). ( E ) The ratio of neuritic to somatic KDEL intensity (N/S). All data are plotted as mean ± s.e.m. ( B–D ) Control: N=3, n=18; Rab10 KD: N=3, n=18. ( B–D ) A one-way ANOVA tested the significance of adding experimental group as a predictor. ****=p<0.0001, ***=p<0.001, **=p<0.01, *=p<0.05.

Article Snippet: Antibody , RTN4 (rabbit polyclonal) , Novus Biologicals , NB100-56681 RRID: AB_838641 , 1:200 (IF).

Techniques: Control, In Vitro, Staining, Marker

Journal: eLife

Article Title: Rab10 regulates neuropeptide release by maintaining Ca 2+ homeostasis and protein synthesis

doi: 10.7554/eLife.94930

Figure Lengend Snippet: ( A ) Typical examples showing the KDEL signals in each condition. Scale bar: 50 μm. ( B ) Quantification of RTN4 intensity in MAP2-positive dendrites. ( C ) The ratio of neuritic to somatic RTN4 intensity (N/S). All data are plotted as mean ± s.e.m. ( B, C ) Control: N=3, n=10; Rab10 KD: N=3, n=11; Rab10 KD+Leu: N=3; n=11. A one-way ANOVA tested the significance of adding experimental group as a predictor. ****=p<0.0001, ns=not significant.

Article Snippet: Antibody , RTN4 (rabbit polyclonal) , Novus Biologicals , NB100-56681 RRID: AB_838641 , 1:200 (IF).

Techniques: Control

Journal: eLife

Article Title: Rab10 regulates neuropeptide release by maintaining Ca 2+ homeostasis and protein synthesis

doi: 10.7554/eLife.94930

Figure Lengend Snippet: Summary of statistical analyses.

Article Snippet: Antibody , RTN4 (rabbit polyclonal) , Novus Biologicals , NB100-56681 RRID: AB_838641 , 1:200 (IF).

Techniques: Control, shRNA, Comparison

Journal: eLife

Article Title: Rab10 regulates neuropeptide release by maintaining Ca 2+ homeostasis and protein synthesis

doi: 10.7554/eLife.94930

Figure Lengend Snippet:

Article Snippet: Antibody , RTN4 (rabbit polyclonal) , Novus Biologicals , NB100-56681 RRID: AB_838641 , 1:200 (IF).

Techniques: Transfection, Construct, shRNA, Control, Recombinant, Software

Journal: bioRxiv

Article Title: Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by curbing luminal transport

doi: 10.1101/2021.05.10.441946

Figure Lengend Snippet: A. Fluorescence micrographs of endogenous RTN4 (immunolabeled) and an exogenously expressed ER luminal marker (mCherry ER ) in fixed COS-7 cells, shown along with a line-scan analysis of the fluorescence intensity along peripheral ER tubules (dashed line in inset). B. Colocalisation analyses of the ER luminal (mCherry ER ) vs overexpressed membrane (Halo-Sec61β or SNAP-RTN4a) markers. Note the high colocalisation of mCherry ER with Sec61β but not with RTN4a. C. Estimated ER tubule diameter in COS-7 cells, transfected with increasing amounts of RTN4a, co-expressed with ER membrane markers (seem methods for details). D. Immunoblot of RTN4 in normal and CRISPR knockout (RTN4 KO) COS-7 and SH-SY5Y cells. E. Measurements as in C of RTN4 KO cell lines. (COS-7 WT; n=47, COS-7 RTN4 KO; n=26, SHSY5Y WT; n=30, SHSY5Y RTN4 KO; n=26). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001, n.s., not significant).

Article Snippet: Rabbit polyclonal antibodies to RTN4 (ab47085) and chicken polyclonal antibodies to MAP2 (ab5392) were obtained from Abcam.

Techniques: Fluorescence, Immunolabeling, Marker, Membrane, Transfection, Western Blot, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by curbing luminal transport

doi: 10.1101/2021.05.10.441946

Figure Lengend Snippet: A. Representative images of intensity photo-activation chase (iPAC) in COS-7 cells transiently expressing RTN3A-Halotag, labelled by tetramethyl rhodamine (TMR) and photoactivatable-Janelia Fluor 646 (paJ646). White box denotes the area of paJ646 photoactivation by laser illumination 405 nm. Traces of photoactivated signal intensity are coloured according to the distance from the photoactivation spot. B. Median halftime of photoactivated signal-rise (arrival t 1/2 , exemplified by vertical dashed lines in A) for ER-targeted photoactivatable GFP (paGFP ER ) or an ER membrane marker (Sec61β-Halo::paJ646), at various distances from the activation spot. Note, measurements for membrane protein mirror simulations of diffusive spread at D = 1.8 μm 2 /s. Luminal protein measurements show transport with super-diffusional scaling and cannot be fit by higher diffusion coefficients (compared to simulations up to D = 6 μm 2 /s, green lines). C. In-silico molecular motion model, simulating diffusive transport of proteins from photoactivation region in the ER tubular network (for a diffusion coefficient, D = 3 μm 2 /s). Simulation results processed identically to experimental data (in A and B), generating curves of local concentration over time, are shown on the right. D. The simulated median arrival t 1/2 plots at various distances from the origin as in B, comparing spread by exclusively by diffusion (red) and active network (blue), which includes randomly oriented flows of velocity ( v = 20 μm/s) in each edge, persisting over τ = 0.1 sec. Note, active flows result in a superdiffusive (sub-quadratic) scaling of signal arrival times versus distance (inset). E. paGFP ER arrival time measurements as in B in mCherry ER (Control) or mCherry-RTN4a overexpressing (RTN4 OE) COS-7 cells (n = 15 each) and F. in RTN4 OE and KO SH-SY5Y cells ( P derived from Kolmogorov-Smirnov test). G. Fluorescence lifetime (FLT) measurements of an ER-localised molecular crowding probe in cells as in F (n = 34, 19, 42, respectively, n.s., not significant).

Article Snippet: Rabbit polyclonal antibodies to RTN4 (ab47085) and chicken polyclonal antibodies to MAP2 (ab5392) were obtained from Abcam.

Techniques: Activation Assay, Expressing, Membrane, Marker, Diffusion-based Assay, In Silico, Concentration Assay, Control, Derivative Assay, Fluorescence

Journal: bioRxiv

Article Title: Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by curbing luminal transport

doi: 10.1101/2021.05.10.441946

Figure Lengend Snippet: A. Representative raw images showing automated detection of paGFP ER transport corresponding to , and . B. Raw images of representative samples (Control and RTN4a OE, corresponding to ). Fluorescence intensity change over time was measured at manually selected ROIs with different distances from the photoactivation spot. Raw data during photoactivation period was fitted to a mono-exponential equation, and the half time to plateau (t 1/2 ) was determined for each distance. C. Analyses as in B in this case comparing WT and RTN4 KO cells (corresponding to ). D. iPAC analysis as in of RTN4 KO COS-7 cells. Note, control signal rise times match well to luminal protein data in . RTN4 KO data results in faster signal spread to long distances, as observed for SH-SY5Y cells in .

Article Snippet: Rabbit polyclonal antibodies to RTN4 (ab47085) and chicken polyclonal antibodies to MAP2 (ab5392) were obtained from Abcam.

Techniques: Control, Fluorescence

Journal: bioRxiv

Article Title: Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by curbing luminal transport

doi: 10.1101/2021.05.10.441946

Figure Lengend Snippet: A. Physical simulation of the dependence between ER Ca 2+ release capacity and luminal transport, incorporating equilibrated binding to Ca 2+ buffer proteins, local ER release, diffusive luminal transport of free/buffered Ca 2+ and luminal flow, with plots of total Ca 2+ released over time. Note, release capacity decreases both as a result of halting active flows and from the direct decrease in flow rate due to tubule narrowing. B. Schema of light-induced ER Ca 2+ release and monitoring assay. C. Representative fluorescence intensity image series of GCaMP3 ER photo-uncaging regions of COS-7 cell preloaded with caged-IP 3 (3 μM, 3 hours, uncaging by continuous 405 nm laser illumination). D. Traces of GCaMP3 ER signal as in C in WT or RTN4a overexpressing (RTN4a OE) SH-SY5Y cells, shown along with the integrated ER Ca 2+ released during uncaging period (the area under the curve, WT; n = 17, RTN4a OE; n = 20). Show are means ± SEM from samples in three independent experiments. **** P < 0.001 (one-way ANOVA). E. ER Ca 2+ release measurements as in D. for WT and RTN4 KO SH-SY5Y cells (WT; n = 15, RTN4 KO; n = 23). * P < 0.05 (student’s t test). F. Fluorescence lifetime imaging microscopy (FLIM) measurements of ER Ca 2+ using the D4ER probe in cells as in E & D. Note Lifetime is inversely proportional to [Ca 2+ ].

Article Snippet: Rabbit polyclonal antibodies to RTN4 (ab47085) and chicken polyclonal antibodies to MAP2 (ab5392) were obtained from Abcam.

Techniques: Binding Assay, Fluorescence, Imaging, Microscopy

Journal: bioRxiv

Article Title: Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by curbing luminal transport

doi: 10.1101/2021.05.10.441946

Figure Lengend Snippet: A. Representative image of differentiated human iPSC-derived cortical neurons (iNeurons) stained by neuron-specific marker MAP2 and β-3-Tubulin. B. Micrographs of immunolabelled endogenous RTN4 in fixed iNeurons with sub-diffraction-limit resolution (nuclei label - Hoechst 33258), and C. of exogenous RTN4a (RTN4a-Halo::TMR) co-stained with a plasma membrane marker (Cellbright). D. Normalised area covered by neurites during outgrowth of WT, RTN4a overexpressing (OE) or knockout (KO) iNeurons, inset: corresponding growth rate extracted from a linear fit. E. Micrographs of iNeurons with TMR-labelled RTN4a-Halo (orange) at the indicated cortical neuron differentiation stage. F. As in E, but in this case RTN4a-Halo was introduced post-differentiation (Day 14). G. Representative time-lapse images of neurite regeneration following a mechanical injury. H. Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments.

Article Snippet: Rabbit polyclonal antibodies to RTN4 (ab47085) and chicken polyclonal antibodies to MAP2 (ab5392) were obtained from Abcam.

Techniques: Derivative Assay, Staining, Marker, Clinical Proteomics, Membrane, Knock-Out

Journal: bioRxiv

Article Title: Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by curbing luminal transport

doi: 10.1101/2021.05.10.441946

Figure Lengend Snippet: Schematic of RTN4-mediated modulation of ER luminal transport with an effect on Ca 2+ distribution and consequently axonal outgrowth/regeneration.

Article Snippet: Rabbit polyclonal antibodies to RTN4 (ab47085) and chicken polyclonal antibodies to MAP2 (ab5392) were obtained from Abcam.

Techniques: