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: 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: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Rtn4 knockdown decreases cell surface localization of integrin β1 and HLA-A. HeLa cells were transiently cotransfected with siRNA against Rtn4 and Block-iT fluorescent control or with Block-iT alone. (A) Cells were immunostained for Rtn4. (B) Rtn4 immunofluorescence intensity was quantified for 30–36 cells per condition. (C) Nonpermeabilized cells were stained for surface-localized integrin β1. (D) Nonpermeabilized cells were stained for surface-localized HLA-A. (E) Integrin β1 surface fluorescence staining intensity was quantified for 39–51 transfected and nontransfected cells per condition. (F) HLA-A surface fluorescence staining intensity was quantified for 34–51 transfected and nontransfected cells per condition. All representative images are maximum intensity projections of confocal z-stacks. Scale bars are 10 µm. Error bars represent SD. ****, p ≤ 0.0001; **, p ≤ 0.01; NS, not significant.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques: Knockdown, Blocking Assay, Control, Immunofluorescence, Staining, Fluorescence, Transfection

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Rtn4a levels modulate the kinetics of VSVG-mCherry trafficking to the cell surface. HeLa cells were transiently cotransfected with a plasmid expressing GFP-NLS as a control, a plasmid expressing Rtn4a-GFP, or siRNA against Rtn4 along with the RUSH construct Str-Ii_VSVG-SBP-mCherry. ER-trapped VSVG was released by addition of 40 µM D-Biotin to the growth media. Cells were fixed before biotin addition and at 15, 30, or 60 min after biotin addition. (A) Representative images of maximum intensity projections of confocal z-stacks showing total VSVG-mCherry. (B) Representative images of single confocal z-planes at the cell periphery to visualize cell surface–localized VSVG-mCherry. (C) Fluorescence intensity of VSVG-mCherry was quantified from the cell surface z-plane and normalized to the total VSVG-mCherry fluorescence intensity measured from the maximum intensity projection of the same cell for 8–12 cells per condition and time point. Scale bars are 10 µm. Error bars represent SD. ****, p ≤ 0.0001; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques: Plasmid Preparation, Expressing, Control, Construct, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Rtn4a levels modulate cell adhesion. HeLa cells were transiently transfected with a plasmid expressing GFP-NLS as a control, a plasmid expressing Rtn4a-GFP, or siRNA against Rtn4. Cells were allowed to attach to collagen-coated dishes for 30 min. Unattached cells were removed, and attached cells were fixed, stained with crystal violet, and lysed. Cell lysate absorbance was measured at 540 nm (see Materials and Methods ). Averages from nine independent experiments are shown. Error bars represent SD. **, p ≤ 0.01; *, p ≤ 0.05.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Staining

Journal: Molecular Biology of the Cell

Article Title: Reticulon 4a promotes exocytosis in mammalian cells

doi: 10.1091/mbc.E19-03-0159

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Rtn4; Aviva Systems Biology (ARP46812_P050) , WB, 1:500.

Techniques:

Journal: Cell Death and Differentiation

Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

doi: 10.1038/cdd.2011.206

Figure Lengend Snippet: Nogo-A expression in cortical neurons. (a) The diagram for antibodies against different antigen recognition sites of human Nogo-A NiR-Δ2 (amino acids (aa) 57–185), NiG-Δ20 (aa 564–749) and Nogo-66 (aa 1055–1120) are the three inhibitory regions of human Nogo-A. TMs are transmembrane domains located on the two sides of Nogo-66. The arrows indicate the target regions of five Nogo-A-specific antibodies, polyclonal antibody (pAb) A201 against aa 201–250, monoclonal antibody (mAb) A563 against aa 563–627, mAb 1E8 against aa 570–691 and pAb A620 against aa 620–1004. 2B3 is targeted to Nogo-66, aa 1055–1120. (b) Top: the expression of neuronal Nogo-A was immunostained with A620 and TuJ1, an immature neuronal marker. Lower: subcellular distribution of neuronal Nogo-A was blotted with A620. Cytosolic (F1), organelle/membrane (F2), nucleic (F3) and cytoskeleton fractions (F4) were the four subcellular fractions of neurons. (c) Living cell staining for neurons were performed with pAb A620, mAb 1E8 or pAb A201 (top). The A620 and 1E8 antibodies had previously been shown to label the extracellular amino-Nogo-A in oligodendrocytes (lower). (d) The total RNA was extracted from neurons exposed to 50 μM hydrogen peroxide (H2O2) for 8 h or not and submitted to quantitative real-time polymerase chain reaction (PCR). β-Actin was selected as an inner standard. (e) Cell lysates from neurons exposed to 50 μM H2O2 for 8 h or not were blotted with pAb A620. β-Actin was selected as an inner standard (left). Quantification by densitometric scans was presented by Nogo-A/β-actin (right). Bar=50 μm. n=3, mean±S.D., paired t-test, *P<0.05, ***P<0.001

Article Snippet: 7 , 15 For western blots, A620 and two commercial antibodies, A201 (Prosci, San Diego, CA, USA; no. 4089) and A563 (BD Biosciences PharMingen, San Diego, CA, USA; no. 612238) were used.

Techniques: Expressing, Marker, Membrane, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

doi: 10.1038/cdd.2011.206

Figure Lengend Snippet: In all, 290–562 residues are the pivotal domain of amino-Nogo-A for resisting to oxidative damage. (a) Schematic diagram of different deletions (M1–M9) of amino-Nogo. (b) Neurons were pretreated with 0.2 μM HIV-1 trans-activating (TAT)-M1-M9 for 2 h, followed by exposure to 50 μM hydrogen peroxide (H2O2) for another 12 h in the presence of TAT-M1-M9. Cell death rate was calculated by propidium iodide (PI) (+)/Hoechst (+). (c) Lysates from neurons treated by H2O2 with or without TAT-AM was performed to western blot with anti-HA and A201 antibodies. β-Actin was selected as a loading control. *Presented the nonspecific bands of anti-HA antibody. (d) HEK293FT transfected with mock or full-length human Nogo-A were treated with indicated concentrations of H2O2 for 1 h, and cell lysates were subjected to western blot probed with A201 antibody. β-Actin was selected as a loading control. The 48 kDa bands were pointed with arrow; n=4, mean±S.D., one-way analysis of variance (ANOVA), *P<0.05; ***P<0.001

Article Snippet: 7 , 15 For western blots, A620 and two commercial antibodies, A201 (Prosci, San Diego, CA, USA; no. 4089) and A563 (BD Biosciences PharMingen, San Diego, CA, USA; no. 612238) were used.

Techniques: Western Blot, Control, Transfection