rtn4a gfp  (New England Biolabs)

Journal: Developmental Cell

Article Title: Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions

doi: 10.1016/j.devcel.2024.05.005

Figure Lengend Snippet: Axonal ER-ribosome interactions are enriched at axon branch points and regulated by extrinsic cues (A–C) Schematic representation of split APEX system used to detect ER-ribosome contacts. When the ER protein Sec61β fused to AP module and the ribosomal protein RpL10A fused to EX module interact with each other, APEX is reconstituted, and contact sites can be visualized as a biotinylation radius around the interactions (A). Representative images of split APEX assay in distal axons from neurons expressing RpL10A-3xHA-EX and V5-AP-Sec61β (left) or V5-AP-RTN4A as a negative control (right). Expression of constructs is visualized with V5 and HA antibodies, and biotinylation is detected with conjugated Strep-555 (B). Quantification of Strep signal in distal axons from neurons as in (B) and without H2O2 as a negative control for the biotinylation reaction (C). (D) Representative image of an axon with branches of a DIV7 neuron expressing V5-AP-Sec61β and RpL10A-3xHA-EX DIV7, showing enrichment of split APEX signal at branch points. White arrowheads indicate branch points. Split APEX signal (Strep555) is shown in magenta and a high-low intensity scale as indicated in lower panel. (E) Quantification of axonal ER-bound ribosomes, using split APEX assay as in (D), analyzed in the axon shaft and at axonal branch points. n = 16 per condition re-analyzed from six independent experiments. ∗ p < 0.05 comparing conditions to each other using Mann-Whitney tests. Scale bars represent 5 μm. (F) Quantification of axonal ER-bound ribosomes, using split APEX assay with RpL10A-3xHA-EX and V5-AP-Sec61β, in neurons stimulated for 30 min with BSA (control), BDNF, NT-3, or NGF. Individual data points each represent a neuron in (C), (E), and (F), and each color represents an independent experiment in (F). Data are presented as mean values ± SEM in (C), (E), and (F). ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001 comparing conditions to control using unpaired t tests (C), Mann-Whitney test (E), or ordinary one-way ANOVA tests (F). Scale bars represent 5 μm (B) and (D). See also Figure S3 and Table S3 .

Article Snippet: RTN4A-GFP , Kind gift from dr. Voeltz (Howard Hughes Medical Institute & Department of Molecular, Cellular and Developmental Biology, University of Colorado) , Addgene #61807.

Techniques: Expressing, Negative Control, Construct, MANN-WHITNEY, Control

Journal: Developmental Cell

Article Title: Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions

doi: 10.1016/j.devcel.2024.05.005

Figure Lengend Snippet:

Article Snippet: RTN4A-GFP , Kind gift from dr. Voeltz (Howard Hughes Medical Institute & Department of Molecular, Cellular and Developmental Biology, University of Colorado) , Addgene #61807.

Techniques: Recombinant, Magnetic Beads, shRNA, Sequencing, Software

Journal: Developmental Cell

Article Title: Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions

doi: 10.1016/j.devcel.2024.05.005

Figure Lengend Snippet: Axonal ER-ribosome interactions are enriched at axon branch points and regulated by extrinsic cues (A–C) Schematic representation of split APEX system used to detect ER-ribosome contacts. When the ER protein Sec61β fused to AP module and the ribosomal protein RpL10A fused to EX module interact with each other, APEX is reconstituted, and contact sites can be visualized as a biotinylation radius around the interactions (A). Representative images of split APEX assay in distal axons from neurons expressing RpL10A-3xHA-EX and V5-AP-Sec61β (left) or V5-AP-RTN4A as a negative control (right). Expression of constructs is visualized with V5 and HA antibodies, and biotinylation is detected with conjugated Strep-555 (B). Quantification of Strep signal in distal axons from neurons as in (B) and without H2O2 as a negative control for the biotinylation reaction (C). (D) Representative image of an axon with branches of a DIV7 neuron expressing V5-AP-Sec61β and RpL10A-3xHA-EX DIV7, showing enrichment of split APEX signal at branch points. White arrowheads indicate branch points. Split APEX signal (Strep555) is shown in magenta and a high-low intensity scale as indicated in lower panel. (E) Quantification of axonal ER-bound ribosomes, using split APEX assay as in (D), analyzed in the axon shaft and at axonal branch points. n = 16 per condition re-analyzed from six independent experiments. ∗ p < 0.05 comparing conditions to each other using Mann-Whitney tests. Scale bars represent 5 μm. (F) Quantification of axonal ER-bound ribosomes, using split APEX assay with RpL10A-3xHA-EX and V5-AP-Sec61β, in neurons stimulated for 30 min with BSA (control), BDNF, NT-3, or NGF. Individual data points each represent a neuron in (C), (E), and (F), and each color represents an independent experiment in (F). Data are presented as mean values ± SEM in (C), (E), and (F). ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001 comparing conditions to control using unpaired t tests (C), Mann-Whitney test (E), or ordinary one-way ANOVA tests (F). Scale bars represent 5 μm (B) and (D). See also Figure S3 and Table S3 .

Article Snippet: Following vectors were used: pSuper, pGW1-mCherry and pGW1-BFP, RTN4A-GFP (a gift from Dr. Gia Voeltz, Addgene plasmid #61807), pEGFP(A206K)-N1 and pEGFP(A206K)-C1 (a gift from Dr. Jennifer Lippincott-Schwartz), GFP-Sec61β (a gift from Dr. Tom Rapoport, Addgene # 15108), RpL10A-tagRFP (a gift from dr. Robert Singer, Addgene #74172), TOM20-V5-FKBP-AP (a gift from Alice Ting, Addgene#120914), mCherry myr 5’/3’-Calreticulin (a gift from dr. Jefferey L. Twiss), Lifeact-mCherry (a gift from dr. Moritoshi Sato, Addgene #67302), HA-KifC1-MD-Strep, DP1-GFP, GFP-SBP-RTN4A, P180-ΔCoiled-coil, P180-Δrepeats-GFP, P180-Coiled-coil and P180-Repeats were previously described.

Techniques: Expressing, Negative Control, Construct, MANN-WHITNEY, Control

Journal: Developmental Cell

Article Title: Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions

doi: 10.1016/j.devcel.2024.05.005

Figure Lengend Snippet:

Article Snippet: Following vectors were used: pSuper, pGW1-mCherry and pGW1-BFP, RTN4A-GFP (a gift from Dr. Gia Voeltz, Addgene plasmid #61807), pEGFP(A206K)-N1 and pEGFP(A206K)-C1 (a gift from Dr. Jennifer Lippincott-Schwartz), GFP-Sec61β (a gift from Dr. Tom Rapoport, Addgene # 15108), RpL10A-tagRFP (a gift from dr. Robert Singer, Addgene #74172), TOM20-V5-FKBP-AP (a gift from Alice Ting, Addgene#120914), mCherry myr 5’/3’-Calreticulin (a gift from dr. Jefferey L. Twiss), Lifeact-mCherry (a gift from dr. Moritoshi Sato, Addgene #67302), HA-KifC1-MD-Strep, DP1-GFP, GFP-SBP-RTN4A, P180-ΔCoiled-coil, P180-Δrepeats-GFP, P180-Coiled-coil and P180-Repeats were previously described.

Techniques: Recombinant, Magnetic Beads, shRNA, Sequencing, Software

Journal: bioRxiv

Article Title: The endoplasmic reticulum adopts two distinct tubule forms

doi: 10.1101/2021.10.26.465969

Figure Lengend Snippet: ( a ) Cell immunolabeled with the sheep anti-Rtn4b antibody. ( b ) Cell immunolabeled with the rabbit anti-Rtn4a/b antibody. The boxed areas in (a,b) correspond to and , respectively.

Article Snippet: Rtn4b-GFP was generated by inserting two PCR-amplified fragments from the initial part (RTN4 AA1-AA185) and the later part (RTN4 AA1005-AA1192 plus AcGFP1) of Rtn4a-GFP into the pcDNA3.1(+) backbone using Gibson Assembly (New England Biolabs, #E2611S).

Techniques: Immunolabeling

Journal: bioRxiv

Article Title: The endoplasmic reticulum adopts two distinct tubule forms

doi: 10.1101/2021.10.26.465969

Figure Lengend Snippet: ( a ) Immunoblotting with the rabbit anti-Rtn4a/b antibody for lysates of the native COS-7 cells (left lane) and COS-7 cells expressing Rtn4a-GFP (right lane). This showed that although the antibody also detects Rtn4a, the major forms in the native COS-7 cell are Rtn4b and Rtn4b2. ( b ) Immunoblotting with the same antibody, for lysates of untransfected U2OS, A7r5, NIH-3T3, and C2C12 cells, showing that Rtn4b and Rtn4b2 are the major forms. ( c ) Immunoblotting with the same antibody, for lysates of cultured primary neurons and astrocytes from the rat hippocampus. ( d ) Enhanced contrast for (c) to visualize the weak signal of Rtn4a, which is presumably present at the neuron plasma membrane.

Article Snippet: Rtn4b-GFP was generated by inserting two PCR-amplified fragments from the initial part (RTN4 AA1-AA185) and the later part (RTN4 AA1005-AA1192 plus AcGFP1) of Rtn4a-GFP into the pcDNA3.1(+) backbone using Gibson Assembly (New England Biolabs, #E2611S).

Techniques: Western Blot, Expressing, Cell Culture