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

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: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Peripheral ER network of U2OS cells expressing GFP-calreticulin from the endogenous promoter in wild type cells. Where indicated, the cells also stably expressed mCherry-tagged wild type ATL-1 or ATL-3 or the corresponding dimerization-defective mutants (ATL-1 R217Q or ATL-3 R213Q). Scale bar = 10 µm. ( B ) As in ( A ), but with cells stably expressing mCherry-tagged ATL-1 or ATL-3 mutants defective in GTP hydrolysis (ATL-1 K80A or ATL-3 K73A). The first and second columns show ATL-1 K80A-expressing cells, in which the ER is either fragmented or converted into long, unbranched tubules. ATL-1 K80A R217Q is both GTPase- and dimerization- defective. The bottom row shows three time points for calreticulin staining. Stationary pixels appear white, while those moving appear in colors. Scale bar = 10 µm. ( C ) As in ( A ), but with cells stably expressing mCherry-tagged cytoplasmic fragments of wild type ATL-1 (cytATL-1 WT), a mutant defective in GTP hydrolysis (cytATL-1 K80A) at low or high level, or a mutant defective in dimerization (cytATL-1 R217Q). CytATL-1 K80A R217Q contains both mutations. The bottom two rows show a close-up image of the peripheral network of the cells depicted in the first two rows. The bottom row shows three time points for calreticulin staining. Stationary pixels appear white, while those moving appear in colors. Scale bar = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.003

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Expressing, Stable Transfection, Staining, Mutagenesis

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Image of a whole U2OS cell expressing GFP-calreticulin. ( B ) As in ( A ), but a cell expressing GFP-ATL-1. ( C ) As in ( A ), but a cell expressing GFP-ATL-3. Scale bar = 10 µm. ( D ) Peripheral ER network in a U2OS cell stably expressing GFP-tagged ATL-2. Scale bar = 10 µm. ( E ) Image of a U2OS cell expressing GFP-ATL-1 K80A, untreated (left panel) or treated with 1 µM nocodazole for 90 min (right panel). Scale bar = 10 µm. ( F ) Peripheral ER network in a U2OS cell stably expressing a GTPase-defective mutant of ATL-2 (GFP-ATL-2 K107A). The right panel shows three time points. Stationary pixels appear white, while those moving appear in colors. Scale bar = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.004

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Expressing, Stable Transfection, Mutagenesis

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Peripheral ER network in a U2OS cell expressing GFP-calreticulin under the endogenous promoter in wild type cells or in cells stably expressing mCherry-tagged Rtn4a. The bottom row shows three time points. Stationary pixels appear white, while those moving appear in colors. Scale bar = 10 µm. ( B ) As in ( A ), but with a higher expression level of mCherry-Rtn4a, resulting in unbranched tubules. ( C ) As in ( B ), but showing a cell with fragmented ER. ( D ) As in ( B ), but with cells that also stably express GFP-ATL-1 or GFP-ATL-3. ( E ) As in ( B ), but with cells that also express a GTPase defective ATL-1 mutant (GFP-ATL-1 K80A). The second column shows three time points for both the ATL and Rtn channels. Stationary pixels appear white, while those moving appear in colors. Scale bar = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.018

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Expressing, Stable Transfection, Mutagenesis

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Extracts of wild type U2OS cells and a Lnp-deleted U2OS clonal cell line were analyzed by immunoblotting with Lnp and Rtn4 antibodies. Equal amounts of total protein were loaded, and GSK3β was used as a loading control. ( B ) Peripheral ER in a U2OS cell lacking Lnp expressing GFP-calreticulin from the endogenous promoter. The cells were fixed and analyzed for GFP fluorescence and for endogenous Rtn4a, using specific antibodies and fluorescently labeled secondary antibodies. Scale bar = 10 µm. ( C ) Peripheral ER in wild type U2OS cells and cells highly expressing Lnp-mCherry. The ER is visualized by GFP-calreticulin expressed under the endogenous promoter. Scale bar = 2 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.022

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Western Blot, Expressing, Fluorescence, Labeling

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Views of wild type U2OS and Lnp-deleted (LnpΔ) cells expressing GFP-calreticulin from the endogenous promoter. LnpΔ cells were generated by CRISPR targeting the start codon of the LNP gene. The bottom row shows magnifications of the boxed areas of the peripheral ER. Scale bars = 10 µm. ( B ) Quantification of the LNP deletion phenotype depicted in A. Wild type and LnpΔ cells were scored blindly for the appearance of peripheral ER. ( C ) Peripheral ER in a LnpΔ cell expressing GFP-calreticulin, as well as stably expressing a low level of Lnp-mCherry. Scale bars = 10 µm. ( D ) As in ( C ), but with cells expressing increasing levels of Lnp-mCherry (left to right). Scale bars = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.021

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Expressing, Generated, CRISPR, Stable Transfection

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Schematic representation of wild type (WT) Lnp and mutants tested for proper localization in the peripheral ER. CC1, CC2, coiled-coil domains 1 and 2, respectively; TM1, TM2, trans-membrane segments 1 and 2, respectively; LNPRK, lunapark motif (single letter code); the segment indicated in red was inserted (extra linker). ( B ) Peripheral ER in U2OS cells expressing GFP-calreticulin under the endogenous promoter in wild type or Lnp-lacking cells (LnpΔ). Where indicated, the cells also stably expressed wild type or mutant Lnp at low levels. Scale bar = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.023

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Expressing, Stable Transfection, Mutagenesis

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Peripheral ER in U2OS cells stably expressing Lnp-mCherry alone or together with GFP-ATL-3 or GFP-ATL-1. Scale bar = 10 micron. ( B ) Peripheral ER in Lnp-lacking U2OS cells expressing GFP-calreticulin. Where indicated, the cells also stably expressed mCherry-tagged ATLs or Rtn4a. Scale bar = 10 micron. DOI: http://dx.doi.org/10.7554/eLife.18605.025

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Stable Transfection, Expressing

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Peripheral ER in U2OS cells stably expressing Lnp-mCherry and GFP-ATL-1 K80A (GTPase defective mutant). The second and fourth rows show three time points. Stationary pixels appear white, while those moving appear in colors. The top five panels depict two entire cells, while the bottom five panels show magnifications of the peripheral ER of these cells. Scale bars = 10 µm. ( B ) Peripheral ER in Lnp-lacking U2OS cells expressing GFP-calreticulin. The cells also stably express mCherry-tagged ATL mutants defective in GTPase activity. The bottom row shows three time points. Scale bar = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.026

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay

Journal: eLife

Article Title: Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

doi: 10.7554/eLife.18605

Figure Lengend Snippet: ( A ) Lnp-Lnp interaction is weakened during mitosis. Purified cytLnp and SBP-tagged cytLnp (cytLnp-SBP) were incubated with interphase cytosol in the absence or presence of cyclin B∆90. The samples were incubated with streptavidin resin, bound material was eluted with biotin, and analyzed by SDS-PAGE and staining with Coomassie blue. The left panels show the input material. ( B ) Purified cytLnp-HA or a phosphomimetic version of it (cytLnp-E-HA) were incubated with Sumo-tagged cytLnp (Sumo-cytLnp) or a phosphomimetic version of it (Sumo-cytLnp-E). Pull-downs were performed with anti-HA resin and the bound material was analyzed by SDS-PAGE and staining with Coomassie blue. ( C ) Lnp residues that were mutated to aspartate or alanine to generate phosphomimetic (S/T → D) or unphosphorylatable (S/T → A) mutants are shown in red. ( D ) Peripheral ER in Lnp-lacking U2OS cells (Lnp-/-) expressing GFP-calreticulin. Where indicated, the cells also stably express at low level mCherry-tagged wild type Lnp, or phosphomimetic or unphosphorylatable mutants. Scale bar = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18605.030

Article Snippet: Primary antibodies used for mammalian cell experiments were: anti-calreticulin from rabbit (Abcam), anti-reticulon 4 from goat (Santa Cruz), anti-Lnp from rabbit (Sigma), HA (Roche), mCherry (polyclonal made in rabbits).

Techniques: Purification, Incubation, SDS Page, Staining, Expressing, Stable Transfection