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: Scientific Reports

Article Title: Axonal branching in lateral olfactory tract is promoted by Nogo signaling

doi: 10.1038/srep39586

Figure Lengend Snippet: ( a ) Immunoblots of Nogo-A, NgR1 and LOTUS in the olfactory bulb at E14, 15, 16, 17, 18 and P0. WT, LOTUS-KO and NgR1-KO indicate protein lysates from wild-type mice at P305, lotus -deficient mice at P237 and ngr1 -deficient mice at P91, respectively. β-actin is used as an internal control protein. ( b ) The expression levels of Nogo-A, NgR1 and LOTUS are quantified by the intensity of each protein immunoblot and normalized to the intensity of β-actin. The significance level was analyzed by performing a Kruskall-Wallis test with Dunn’s multiple comparison analysis. (Nogo-A: n = 4 experiments; LOTUS: n = 3 experiments; NgR1: n = 3 experiments).

Article Snippet: The monoclonal mouse antibody against Nogo-A (Millipore, Temecula, CA, USA), polyclonal goat antibody against mouse NgR1 (R&D Systems, Minneapolis, MN, USA), polyclonal rabbit antibody against βIII-tubulin (Genscript, Piscataway, NJ, USA), monoclonal mouse antibody against Tau-1 (Millipore), polyclonal rabbit antibody against Tau-1 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO, USA), biotinylated goat antibody against hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated goat antibody against hamster IgG (Jackson ImmunoResearch), biotinylated donkey antibody against goat IgG (Jackson ImmunoResearch), Alexa488-labeled goat antibody against mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa488-labeled donkey antibody against guinea pig IgG (Jackson ImmunoResearch), Alexa594-labeled goat antibody against mouse IgG (Jackson ImmunoResearch), Alexa647-labeled goat antibody against rabbit IgG (Invitrogen), horseradish peroxidase (HRP)-conjugated donkey antibody against rabbit IgG (GE Healthcare), HRP-conjugated sheep antibody against mouse IgG (GE Healthcare, Chicago, IL, USA), HRP-conjugated donkey antibody against goat IgG (Jackson ImmunoResearch) and HRP-conjugated donkey antibody against mouse IgM (Jackson ImmunoResearch) were obtained commercially.

Techniques: Western Blot, Control, Expressing, Comparison

Journal: Scientific Reports

Article Title: Axonal branching in lateral olfactory tract is promoted by Nogo signaling

doi: 10.1038/srep39586

Figure Lengend Snippet: ( a1,b1 ) Immunostaining of βIII-tubulin, a neuronal marker. ( a2,b2 ) Immunostaining of Tau-1, an axonal marker in the same cells of (1), ( a3,b3 ) merged images of (1) and (2), ( a4,b4 ) and immunostaining of Nogo-A in pseudo-color. ( a5,b5 ) Dashed boxes in images of a3 and b3 correspond to images of a5 and b5 at higher magnification, respectively. Arrowheads indicate axonal collateral branches. Dissociated OB neurons from E14.5 wild type mice (NgR1+/+) ( a ) or ngr1 -deficient mice (NgR1−/−) ( b ) were cultured for 7days. Branching points in ngr1 -deficient (NgR1−/−) mice were decreased in comparison with the wild type. ( c ) Quantitative analysis of axonal branching points in cultured OB neurons. Significance, indicated by (*), was obtained by performing Student’s unpaired t-test. *is P = 0.0437 (LOTUS+/+ versus LOTUS−/− (n = 4)), ** is P = 0.0029 (NgR1+/+ versus NgR1−/− (n = 3)), and comparisons indicated by (†) were obtained by performing a one-way ANOVA test with all pairwise multiple comparisons test (Tukey’s test). † is P < 0.05. †† is P < 0.000001. Scale bars: 50 μm.( d ) Quantitative analysis of axon lengths in cultured OB neurons. Axon lengths of Tau-1-positive neurites were measured using NIH ImageJ software. Nogo-A knockdown (sh-Nogo, using sh-1 in LOTUS+/+ and sh-3 in LOTUS−/−) resulted in the increase of axon length. Similarly, axon lengths in ngr1 -deficient (NgR1−/−) mice were increased compared to those in the wild-type mice. Significance was obtained by performing one-way ANOVA with Holm-Sidak’s multiple comparison test. * P = 0.0192 (control versus sh-Nogo treatment (n = 17–34 axons)); ** P = 0.0218 (wild type versus NgR1−/− (n = 4 littermates)); *** P = 0.0229 (wild type versus LOTUS−/− (n = 5 littermates)).

Article Snippet: The monoclonal mouse antibody against Nogo-A (Millipore, Temecula, CA, USA), polyclonal goat antibody against mouse NgR1 (R&D Systems, Minneapolis, MN, USA), polyclonal rabbit antibody against βIII-tubulin (Genscript, Piscataway, NJ, USA), monoclonal mouse antibody against Tau-1 (Millipore), polyclonal rabbit antibody against Tau-1 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO, USA), biotinylated goat antibody against hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated goat antibody against hamster IgG (Jackson ImmunoResearch), biotinylated donkey antibody against goat IgG (Jackson ImmunoResearch), Alexa488-labeled goat antibody against mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa488-labeled donkey antibody against guinea pig IgG (Jackson ImmunoResearch), Alexa594-labeled goat antibody against mouse IgG (Jackson ImmunoResearch), Alexa647-labeled goat antibody against rabbit IgG (Invitrogen), horseradish peroxidase (HRP)-conjugated donkey antibody against rabbit IgG (GE Healthcare), HRP-conjugated sheep antibody against mouse IgG (GE Healthcare, Chicago, IL, USA), HRP-conjugated donkey antibody against goat IgG (Jackson ImmunoResearch) and HRP-conjugated donkey antibody against mouse IgM (Jackson ImmunoResearch) were obtained commercially.

Techniques: Immunostaining, Marker, Cell Culture, Comparison, Software, Knockdown, Control

Journal: Scientific Reports

Article Title: Axonal branching in lateral olfactory tract is promoted by Nogo signaling

doi: 10.1038/srep39586

Figure Lengend Snippet: ( a ) Schematic drawing of the lateral view of axonal branching in the LOT. The dashed square indicates the position of branch point analysis. ( b–e ) Lateral views of LOT axonal branching in whole brain from lotus -deficient mice (LOTUS−/−) and ngr1 -deficient (NgR1−/−) mice at E18. Compared to wild-type mice ( b ), the axonal branching points of the LOT were increased (white arrowheads) in LOTUS−/− ( c ), whereas the branching points in NgR1−/− were decreased ( d ). Abnormally increased axonal branching observed in LOTUS−/− was rescued in double homozygous mutants of lotus and ngr1 (LOTUS−/−; NgR1−/−) ( e ). Scale bar: 50 μm. ( f–h ) Quantitative analysis of branching points in the LOT. ( f ) Comparison of wild-type, LOTUS+/− and LOTUS−/− mice. ( g ) Comparison of wild-type, NgR1+/− and NgR1−/− mice. ( h ) Comparison of wild-type, NgR1+/− and NgR1−/− mice in the background of LOTUS−/− mice. Significance, indicated by (*), was analyzed using a Tukey’s one-way ANOVA test. ( f ) * is P = 0.031 (LOTUS+/+ versus LOTUS−/− (n = 5 littermates)). ( g ) ** is P = 0.00008 (NgR1+/+ versus NgR1−/− (n = 4 littermates)) and *** is P = 0.0016 (NgR1+/− versus NgR1−/− (n = 4 littermates)). ( h ) **** is P = 0.0001 (LOTUS−/−; NgR1+/+ versus LOUTS−/−; NgR1−/− (n = 3 littermates)) and ***** is P = 0.0076 (LOTUS−/−; NgR1+/+ versus LOTUS−/−; NgR1+/− (n = 3 littermates)).

Article Snippet: The monoclonal mouse antibody against Nogo-A (Millipore, Temecula, CA, USA), polyclonal goat antibody against mouse NgR1 (R&D Systems, Minneapolis, MN, USA), polyclonal rabbit antibody against βIII-tubulin (Genscript, Piscataway, NJ, USA), monoclonal mouse antibody against Tau-1 (Millipore), polyclonal rabbit antibody against Tau-1 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO, USA), biotinylated goat antibody against hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated goat antibody against hamster IgG (Jackson ImmunoResearch), biotinylated donkey antibody against goat IgG (Jackson ImmunoResearch), Alexa488-labeled goat antibody against mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa488-labeled donkey antibody against guinea pig IgG (Jackson ImmunoResearch), Alexa594-labeled goat antibody against mouse IgG (Jackson ImmunoResearch), Alexa647-labeled goat antibody against rabbit IgG (Invitrogen), horseradish peroxidase (HRP)-conjugated donkey antibody against rabbit IgG (GE Healthcare), HRP-conjugated sheep antibody against mouse IgG (GE Healthcare, Chicago, IL, USA), HRP-conjugated donkey antibody against goat IgG (Jackson ImmunoResearch) and HRP-conjugated donkey antibody against mouse IgM (Jackson ImmunoResearch) were obtained commercially.

Techniques: Comparison