Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: Interactions between tubule-forming proteins and FIT2. (A) IP of FIT2-HA in WT and FIT2-HA KI HepG2 cells. WT and FIT2-HA KI cells were lysed in 1% digitonin-containing buffer. IP was performed with anti-HA antibodies. The samples were analyzed by immunoblotting (IB) with the indicated antibodies. (B) coIP of FIT2 and ER tubule-forming proteins in HepG2 cells. IP was performed as in A. Samples were analyzed by IB with antibodies of different ER membrane proteins. (C) FIT2-HA and REEP5-Myc were cotransfected into HEK293T cells and solubilized in triton-containing buffer or transfected individually into cells in different dishes, followed by mixing of the Triton-solubilized cell extracts. IP was performed with anti-HA or anti-Myc antibodies. (D) As in C, but with cells expressing FIT2-HA and/or GFP-Rtn4a. The asterisk (*) indicates degraded GFP-Rtn4a. (E) coIP of FIT2 truncations and Rtn4/REEP5. HEK293T cells expressing FIT2-HA, FIT2 truncations, or empty vector were lysed in 1% Triton-containing buffer and cell lysates immunoprecipitated with anti-HA antibodies.

Article Snippet: The primary antibodies used for immunoblotting are mouse–anti-GAPDH (Proteintech; 60004–1), rabbit–anti-NOGO (Rtn4; Novus; NB100-5681), rabbit–anti-REEP5 (Proteintech; 14643–1-AP), rabbit–anti-calnexin (Proteintech; 10427–2-AP), rabbit–anti-GFP (Proteintech; 50430–2-AP), rabbit–anti–septin 7 (Abcam; ab175229), rabbit–anti–septin 6 (Immunoway; YT4205), rabbit–anti-MSF (septin 9; Abcam; ab114099), mouse–anti-His (MBL; D291-3), mouse–anti–β-actin (abgent; AM1021B), rabbit–anti-BIP (Abcam; ab21685), mouse–anti-PDI (Abcam; ab2792), mouse–anti-ACSL3 (Abnova; H00002181-B01P), mouse–anti-BSCL2 (seipin; Abnova; H00026580-A02), and rabbit–anti-sec61β (Abcam; ab78276).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: Purification of septins and FIT2. (A) Representation of the septin2/6/7 hexamer. (B) Purified septin2/6/7 hexamer was detected by SDS-PAGE and Coomassie blue staining. The strep tag on the C-terminus of septin 7 was cleaved by thrombin. (C) The purified FIT2-strep-His8-GFP fusion protein was detected by SDS-PAGE and Coomassie blue staining. (D) Representation of FIT2 proteoliposomes. (E) Purified His-tagged septins 2, 6, 7, and 9 were detected by SDS-PAGE and Coomassie blue staining. (F) Purified His-tagged septin 7 CC and septin 7ΔCC were detected by SDS-PAGE and Coomassie blue staining. (G–I) As in but with COS-7 cells depleted of Sec61β. n = 51–88 cells/group. Mann–Whitney test; NS, P > 0.05. Scale bar, 10 µm. Error bars represent SEM. (J) As in , Sec61β and GAPDH were detected by immunoblotting (IB). (K) coIP of FIT2 and Rtn4/REEP5 before and after OA treatment. WT and FIT2-HA KI HepG2 cells were delipidated by starving for 60 h or treating with OA for 15 min and lysed in 1% digitonin buffer. IP was performed with anti-HA antibodies, and samples were analyzed by immunoblotting. The relative amount of Rtn4 (coIP) or REEP5 (coIP) compared with FIT2-HA (IP) was quantified by Gel-Pro analyzer software. siSec61β, small interfering Sec61β.

Article Snippet: The primary antibodies used for immunoblotting are mouse–anti-GAPDH (Proteintech; 60004–1), rabbit–anti-NOGO (Rtn4; Novus; NB100-5681), rabbit–anti-REEP5 (Proteintech; 14643–1-AP), rabbit–anti-calnexin (Proteintech; 10427–2-AP), rabbit–anti-GFP (Proteintech; 50430–2-AP), rabbit–anti–septin 7 (Abcam; ab175229), rabbit–anti–septin 6 (Immunoway; YT4205), rabbit–anti-MSF (septin 9; Abcam; ab114099), mouse–anti-His (MBL; D291-3), mouse–anti–β-actin (abgent; AM1021B), rabbit–anti-BIP (Abcam; ab21685), mouse–anti-PDI (Abcam; ab2792), mouse–anti-ACSL3 (Abnova; H00002181-B01P), mouse–anti-BSCL2 (seipin; Abnova; H00026580-A02), and rabbit–anti-sec61β (Abcam; ab78276).

Techniques: Purification, SDS Page, Staining, Strep-tag, MANN-WHITNEY, Western Blot, Software

Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: Tubule-forming proteins are required in the maintenance of LD homeostasis in HepG2 cells. (A) The levels of FIT2 and FIT2-related tubule-forming proteins were determined by Western blotting or real-time PCR as indicated after HepG2 cells were transfected with siRNAs for 48 h. (B) Western blotting of Rtn4 in WT and Rtn4-KO HepG2 cells. (C) LipidTOX staining of LDs and quantification of number of LDs per cell, as well as LD size, in WT and Rtn4 KO HepG2 cells without (−OA) or with (+OA) OA treatment. Colorized pictures were acquired using Imaris software. Different colors indicate different LD diameters, as labeled. n = 50–76 cells/group, total LD numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. (D) The level of Climp-63 was determined by Western blotting after HepG2 cells were transfected with the indicated siRNAs for 48 h. (E) As in C, except in Control and Climp-63–depleted HepG2 cells. For groups without OA treatment, n = 76 and 74 cells, total LD numbers >2,000. For groups with OA treatment, n = 45 and 51 cells, total LD numbers >2,000. Mann–Whitney test; NS, P > 0.05. Scale bar, 10 µm. (F) Levels of LD-related proteins in HepG2 cells transfected with the indicated siRNAs. (G) Septin 2 KO (SEPT2 KO) HepG2 cells were generated using CRISPR/Cas9, two single clones (#10 and #16) picked, and the protein level of septin 2 in WT and SEPT2 KO cells measured by immunoblotting (IB). (H–J) The knockdown efficiencies of siRNAs targeting septin 6, 7, and 9 were tested by Western blotting. (K) As in , LipidTOX staining and Imaris 3D images of OA-treated HepG2 cells transfected with the indicated siRNAs. The numbers and sizes of the LDs in each group were determined by Imaris surface analysis. n = 50–76 cells/group, n = 74–101 cells/group, total LD numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. siClimp-63, small interfering Climp-63; siControl, small interfering Control; si FIT2, small interfering siFIT2; siREEP5, small interfering REEP5; siRtn4, small interfering Rtn4; siSEPT, small interfering SEPT. Error bars represent SEM.

Article Snippet: The primary antibodies used for immunoblotting are mouse–anti-GAPDH (Proteintech; 60004–1), rabbit–anti-NOGO (Rtn4; Novus; NB100-5681), rabbit–anti-REEP5 (Proteintech; 14643–1-AP), rabbit–anti-calnexin (Proteintech; 10427–2-AP), rabbit–anti-GFP (Proteintech; 50430–2-AP), rabbit–anti–septin 7 (Abcam; ab175229), rabbit–anti–septin 6 (Immunoway; YT4205), rabbit–anti-MSF (septin 9; Abcam; ab114099), mouse–anti-His (MBL; D291-3), mouse–anti–β-actin (abgent; AM1021B), rabbit–anti-BIP (Abcam; ab21685), mouse–anti-PDI (Abcam; ab2792), mouse–anti-ACSL3 (Abnova; H00002181-B01P), mouse–anti-BSCL2 (seipin; Abnova; H00026580-A02), and rabbit–anti-sec61β (Abcam; ab78276).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Staining, Software, Labeling, MANN-WHITNEY, Generated, CRISPR, Clone Assay

Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: FIT2-interacting proteins in LD biogenesis. (A) LipidTOX staining of LDs and quantification of LDs per cell, as well as LD size, in control and tubule-forming protein-depleted HepG2 cells without OA treatment. 3D pictures of LDs were acquired under a confocal microscope by serial layer scanning along the z axis. Colorized pictures were acquired using Imaris software. Different colors indicate different LD diameters, as labeled. n = 86–200 cells/group. All graphs are representative of three repetitions, total LDs numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. (B) As in A, except each group of cells was treated with 0.2 mM OA for 6 h before LipidTOX staining. n = 82–143 cells/group, total LDs numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. (C and D ) As in A and B, LDs were stained in WT (SEPT2-WT) and septin 2 KO (SEPT2-KO) HepG2 cells with (C) or without (D) OA treatment. The numbers and sizes (diameter) of LDs were measured. For groups without OA treatment, n = 46–76 cells/group; for groups with OA treatment, n = 53–78 cells/group, total LD numbers >2,000. Mann–Whitney test; **, P < 0.01; ***, P < 0.001. Scale bar, 10 µm. siControl, small interfering Control; siFIT2, small interfering FIT2; siREEP5, small interfering siREEP5; siRtn4, small interfering Rtn4. Error bars represent SEM.

Article Snippet: The primary antibodies used for immunoblotting are mouse–anti-GAPDH (Proteintech; 60004–1), rabbit–anti-NOGO (Rtn4; Novus; NB100-5681), rabbit–anti-REEP5 (Proteintech; 14643–1-AP), rabbit–anti-calnexin (Proteintech; 10427–2-AP), rabbit–anti-GFP (Proteintech; 50430–2-AP), rabbit–anti–septin 7 (Abcam; ab175229), rabbit–anti–septin 6 (Immunoway; YT4205), rabbit–anti-MSF (septin 9; Abcam; ab114099), mouse–anti-His (MBL; D291-3), mouse–anti–β-actin (abgent; AM1021B), rabbit–anti-BIP (Abcam; ab21685), mouse–anti-PDI (Abcam; ab2792), mouse–anti-ACSL3 (Abnova; H00002181-B01P), mouse–anti-BSCL2 (seipin; Abnova; H00026580-A02), and rabbit–anti-sec61β (Abcam; ab78276).

Techniques: Staining, Microscopy, Software, Labeling, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: Functional tests of FIT2-interacting proteins. (A) Representative confocal images of wild-type and yop-1(ok3629) larval stage L4 animals carrying the ldrIs2 (Pmdt28::mdt28::cherry) transgene. Images are 3D projections of 2.62-µm confocal z -stacks that covered the posterior body forward from the vent. LD quantification is shown on the right. n = 16 ( wild-type ) and 18 ( yop-1 ) animals. Unpaired t test, ***, P < 0.001. Scale bar, 10 µm. (B) As in A, but with RET-1–depleted animals generated by RNAi feeding. n = 7 ( wild-type ) and 11 ( ret-1 ) animals. Unpaired t test, ***, P < 0.001. Scale bar, 10 µm. (C) As in A, but with unc-61(e228) mutant. n = 10 ( wild-type ) and 13 ( unc-61 ) animals. Unpaired t test, ***, P < 0.001. Scale bar, 10 µm. Error bars represent SEM. (D) Differentiated 3T3-L1 cells were collected at the indicated times, and protein levels were detected by immunoblotting (IB) with the indicated antibodies. (E) The knockdown efficiency of the indicated siRNAs in 3T3-L1 cells was measured by IB. (F) Representative images of 3T3-L1 adipocytes. On day 10, differentiated 3T3-L1 cells were fixed and stained with Oil Red. Images were captured using a stereomicroscope under the indicated magnifications. Scale bar, 2 mm. siControl, small interfering Control; simSEPT2: small interfering mouse SEPT2; simREEP5: small interfering mouse REEP5; simRtn4, small interfering mouse Rtn4.

Article Snippet: The primary antibodies used for immunoblotting are mouse–anti-GAPDH (Proteintech; 60004–1), rabbit–anti-NOGO (Rtn4; Novus; NB100-5681), rabbit–anti-REEP5 (Proteintech; 14643–1-AP), rabbit–anti-calnexin (Proteintech; 10427–2-AP), rabbit–anti-GFP (Proteintech; 50430–2-AP), rabbit–anti–septin 7 (Abcam; ab175229), rabbit–anti–septin 6 (Immunoway; YT4205), rabbit–anti-MSF (septin 9; Abcam; ab114099), mouse–anti-His (MBL; D291-3), mouse–anti–β-actin (abgent; AM1021B), rabbit–anti-BIP (Abcam; ab21685), mouse–anti-PDI (Abcam; ab2792), mouse–anti-ACSL3 (Abnova; H00002181-B01P), mouse–anti-BSCL2 (seipin; Abnova; H00026580-A02), and rabbit–anti-sec61β (Abcam; ab78276).

Techniques: Functional Assay, Generated, Mutagenesis, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: FIT2-interacting proteins in early steps of LD formation. (A) Representative images of remaining LDs after delipidation and nascent LDs formed after OA treatment in control and ER tubule-forming protein–depleted COS-7 cells. Each group of cells expressing LiveDrop was delipidated for 60 h and then treated with 0.2 mM OA for 15 min to induce nascent LD formation. Quantification of LDs is shown on the right. n = 36–42 cells/group. Unpaired t test; NS, P > 0.05. Scale bar, 10 µm. (B) As in A, but with cells overexpressing Flag–Climp-63. n = 33–37 cells/group. One-way ANOVA, ***, P < 0.001. Scale bar, 10 µm. (C) As in A, but nascent LDs in septin 2 knockdown COS-7 cells were counted. The bottom images show enlargements of the boxed regions over the tubular ER network. n = 13–16 cells/group. Unpaired t test, ***, P < 0.001. Scale bar, 10 µm; 3 µm (inset). Error bars represent SEM. (D) Time-lapse stills of nascent LD formation in control or septin 2 knockdown COS-7 cells. The histogram on the right shows the quantification of the frequency of nascent LDs in the indicated time frame. n = 3 or 4 cells/group. Scale bar, 5 µm. siControl, small interfering Control; siFIT2, small interfering FIT2; siREEP5, small interfering siREEP5; siRtn4, small interfering Rtn4; siSEPT, small interfering SEPT.

Article Snippet: The primary antibodies used for immunoblotting are mouse–anti-GAPDH (Proteintech; 60004–1), rabbit–anti-NOGO (Rtn4; Novus; NB100-5681), rabbit–anti-REEP5 (Proteintech; 14643–1-AP), rabbit–anti-calnexin (Proteintech; 10427–2-AP), rabbit–anti-GFP (Proteintech; 50430–2-AP), rabbit–anti–septin 7 (Abcam; ab175229), rabbit–anti–septin 6 (Immunoway; YT4205), rabbit–anti-MSF (septin 9; Abcam; ab114099), mouse–anti-His (MBL; D291-3), mouse–anti–β-actin (abgent; AM1021B), rabbit–anti-BIP (Abcam; ab21685), mouse–anti-PDI (Abcam; ab2792), mouse–anti-ACSL3 (Abnova; H00002181-B01P), mouse–anti-BSCL2 (seipin; Abnova; H00026580-A02), and rabbit–anti-sec61β (Abcam; ab78276).

Techniques: Expressing

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:

Journal: Neuroscience

Article Title: Nogo presence is inversely associated with shifts in cortical microglial morphology following experimental diffuse brain injury

doi: 10.1016/j.neuroscience.2017.07.027

Figure Lengend Snippet: Representative images of DAB labelling for Nogo receptor (NgR). NgR was observed as punctate staining within processes and cell bodies; these cells were most likely neurons. Maximal staining was observed at day 1 post-injury.

Article Snippet: Sections were then incubated at 4ºC overnight in 1% blocking solution containing either rabbit anti-Iba-1 primary antibody (Wako, cat #019-19741, 1:5000) or rabbit anti-Nogo receptor (Alomone Labs, cat #ANT-008, 1:500).

Techniques: Staining