Fig 1: RTN3 over-expression induces ER tubules fragmentation during starvation.(A, B) Schematic representation of the protein structure and topology of FAM134B (A) and RTN3 long and short isoforms (B). (C) Immunofluorescence of HA and LC3B in U2OS TRex stable cell lines expressing FLAG-HA-RTN3L after 24 hr treatment with 1 µg/ml of doxycycline. Cells were kept in standard growing condition (DMEM with 10% FBS) or starved with EBSS for 6 hr. RTN3L was monitored using anti HA antibody, while autophagy induction was visualized using anti-LC3B antibody. Bafilomycin A1 was added at a final concentration of 200 ng/ml. Scale bars: 10 µm. (D) Quantification of U2OS TRex FLAG-HA-RTN1-4L cells with ER tubule fragment after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Number of cells >100 for each condition. Data are representative of three independent biological replicates. Error bars indicate s.d. (E) Super-resolution fluorescence microscopy (dSTORM) of ER fragments in U2OS TRex FLAG-HA-RTN3L cells stained with anti HA and anti LC3B antibodies after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Scale bar: 0.5 µm. (F) Schematic representation of ER tubules fragmentation and LC3 labeling in U2OS TRex FLAG-HA-RTN3L cells. The red square indicates the level of high resolution represented in panel E.DOI: http://dx.doi.org/10.7554/eLife.25555.002
Fig 2: RTN3 interacts with the autophagy modifiers.(A) Venn diagrams of the interactors of the four RTNs. Numbers represent the identified peptides significantly enriched in three IP and mass spectrometry replicates for each RTN. (B) Annotation enrichment analysis of the interactors of long RTN1-4 isoforms. Bars represent the significantly enriched gene ontology biological process (GOBP), the gene ontology cellular components (GOCC), the gene ontology molecular function (GOMF), the over-expressed pathways (KEGG) and the domain enrichment (Pfam). The numeric value on the right side of the bar shows the Benjamini-Hochberg FDR value. (C) Scatter-plot for 1D annotation enrichment analysis of RTN3L interactor partners significantly enriched in three different IPs. (D) Volcano-plot for RTN3L SILAC-based interactome. Peptides with and Log2 Ratio H/L ≥1 and –Log10 p value > 1.3 are labeled in red. Three biological replicates were analyzed. (E) Co-IP of endogenous GABARAP and GABARAP-L1 with over-expressed long and short isoforms of RTN1-4. Over-expression was induced for 24 hr in U2OS TRex stable cell lines using 1 µg/ml of doxycycline. Bafilomycin A1 was added at the final concentration of 200 ng/ml for 2 hr. (F) Endogenous Co-IP of RTN3 with GABARAP in A549 cells. The ‘empty’ lane represents unconjugated beads. Bafilomycin A1, 200 ng/ml, was added for 2 hr.DOI: http://dx.doi.org/10.7554/eLife.25555.01910.7554/eLife.25555.020Figure 5—source data 1.IP-interactome of RTN1, RTN2, RTN3 and RTN4 long isoforms.IP-interactome analyses were performed using the SILAC-labeling strategy in U2OS after 24-hr treatment with 1 µg/ml of doxycycline. Bafilomycin A1, 200 ng/ml, was added for 2 hr. Peptides with Log2 (Heavy/Light [H/L]) ratios ≥1 and a p value ≤ 0.05 were considered significantly enriched.DOI: http://dx.doi.org/10.7554/eLife.25555.02010.7554/eLife.25555.021Figure 5—source data 2.IP-interactome of RTN1, RTN2, RTN3 and RTN4 short isoforms.IP-interactome analyses were performed using the SILAC labeling strategy in U2OS after 24-hr treatment with 1 µg/ml of doxycycline. Bafilomycin A1, 200 ng/ml, was added for 2 hr. Peptides with Log2 (Heavy/Light [H/L]) ratios ≥ 1 and a p value ≤ 0.05 were considered significantly enriched.DOI: http://dx.doi.org/10.7554/eLife.25555.021
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