Journal: Molecular Cancer

Article Title: MiR-124 suppresses tumor growth and metastasis by targeting Foxq1 in nasopharyngeal carcinoma

doi: 10.1186/1476-4598-13-186

Figure Lengend Snippet: The ectopic miR-124 induced the expression of Foxq1 by directly targeting its 3 ′ -UTR. A , Putative miR-124 binding site in the 3′-UTR region of Foxq1 and interspecies conservation of seed matching sequences (gray box). (B and C) , The gene expression of Foxq1 in NPC cells compared with NP69 cells by RT-qPCR and western bolt. D , The protein expression level of Foxq1 in lv-miR-124/5-8 F cell and lv-miR-124/6-10B cell compared with control. E , Diagram of wt 3′-UTR and mut 3′-UTR of Foxq1 contained reporter constructs. F , Luciferase reporter assays in 5-8 F cells, co-transfected of wt/mut 3′-UTR with miRNAs as indicated. Statistical analysis was performed using the t-tests. The data represent the mean values of three independent experiments. *, P < 0.05, **, P < 0.01.

Article Snippet: Then, the membrane was incubated with goat polyclonal antibody against human Foxq1 (WuXi PharmaTech Cayman China) followed by HRP (horseradish peroxidase)-labeled goat-anti mouse IgG (Santa Cruz Biotechnology) and detected by chemiluminescence.

Techniques: Expressing, Binding Assay, Gene Expression, Quantitative RT-PCR, Western Blot, Control, Construct, Luciferase, Transfection

Journal: Molecular Cancer

Article Title: MiR-124 suppresses tumor growth and metastasis by targeting Foxq1 in nasopharyngeal carcinoma

doi: 10.1186/1476-4598-13-186

Figure Lengend Snippet: The suppression of down-regulated of FOXQ1 was consistent with the suppression of the ectopic miR-124 and overexpression of FOXQ1 could rescue partially the suppression of miR-124. A , 5-8 F cells were transfected with siRNA-Foxq1 or miR-124 mimics. Effect of siRNA-Foxq1 or miR-124 on cell proliferation was measured by CCK-8 assay in 5-8 F lines. (B and C) , The migrated and invasive cell numbers of NPC cells. D , The protein expression level of Foxq1 was detected after transfect with siRNA-Foxq1 or miR-124 mimics. (E and F), Effect of over-expression regulated Foxq1 in lv-miR-124/5-8 F cells on cell proliferation and tablet cloning ability were measured. (G and H), Effect of up-regulated Foxq1 in lv-miR-124/5-8 F cells on cell migration and invasion were test. (I and J) , Stable expression of Foxq1 in lv-miR-124/5-8 F cells (lv-Foxq1/lv-miR-124/5-8 F) was constructed. Statistical analysis was performed using the t-tests. The data represent the mean values of three independent experiments. **, P < 0.01.

Article Snippet: Then, the membrane was incubated with goat polyclonal antibody against human Foxq1 (WuXi PharmaTech Cayman China) followed by HRP (horseradish peroxidase)-labeled goat-anti mouse IgG (Santa Cruz Biotechnology) and detected by chemiluminescence.

Techniques: Over Expression, Transfection, CCK-8 Assay, Expressing, Cloning, Migration, Construct

Journal: Molecular Cancer

Article Title: MiR-124 suppresses tumor growth and metastasis by targeting Foxq1 in nasopharyngeal carcinoma

doi: 10.1186/1476-4598-13-186

Figure Lengend Snippet: Mir-124 and Foxq1 are inversely correlated in NPC tissues. A , The average expression level of Foxq1 in human NPC specimens compared with non-cancer biopsy samples. B , The Foxq1 expression of clinicalIstage had lower expression than in the IVstage. C , Foxq1 expression was lower in stage T1, whereas stages T2-T4 had higher levels. D , Representative IHC for Foxq1 in non-cancer biopsy samples and NPC specimen with different clinical stages. E , In the mRNA levels, a significant inverse correlation was observed after correlated Foxq1 with the miR-124 expression levels in the 178 NPC specimens. F , Statistical quantification of the IHC scores of Foxq1 between non-cancer biopsy samples and NPC specimen with different clinical stages. Scale bars, 100 μm. G , A scatter diagram shows an inverse correlation between miR-124 and Foxq1 expression in the same set of NPC tissue (Spearman’s correlation analysis, r = -0.6056; p < 0.0001).Statistical analysis was performed using the nonparametric tests (A) and the one-way ANOVA (B, C, E, F) . *, P < 0.05, **, P < 0.01.

Article Snippet: Then, the membrane was incubated with goat polyclonal antibody against human Foxq1 (WuXi PharmaTech Cayman China) followed by HRP (horseradish peroxidase)-labeled goat-anti mouse IgG (Santa Cruz Biotechnology) and detected by chemiluminescence.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: NCAM2 and NCAM1 are BACE1 substrates in the mouse olfactory bulb. A–B , schematic diagram of the two mouse NCAM2 (transmembrane [TM] and GPI-anchored) isoforms ( A ) and three major mouse NCAM1 (transmembrane [NCAM1-180 and NCAM1-140] and GPI-anchored [NCAM1-120]) isoforms ( B ) resulting from alternative splicing. NCAM2 and NCAM1 have five immunoglobulin (Ig)-like domains represented by ovals and two fibronectin type III repeats (FN) represented by rectangles. C , schematic presentation of NCAM2-TM and polysialylated NCAM1-140 illustrating antibody-binding sites. NCAM2 contains eight putative Asn-linked glycosylation sites (N-glycosylation) indicated by blue triangles, while NCAM1 has six N-glycosylation sites. In the fifth Ig (Ig5) of NCAM1, two of three N-glycosylation sites are polysialylated, represented by a chain of green circles (sialic acid). D , representative immunoblot of PBS soluble fraction (Soluble) and membrane fraction (Membrane) of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using anti-NCAM2 (sc-136328), anti-NCAM1 (AF-2408), BACE1 (D10E5), GAPDH (MAB374), and anti-calnexin (610523) antibodies. BACE1-specific soluble NCAM2 (sNCAM2β) and NCAM1 (sNCAM1β) are observed in BACE1+/+ mice, but not in BACE1−/− mice. Full-length NCAM1 (NCAM1-180 and NCAM1-140, but not NCAM1-120) levels were slightly increased in BACE1−/−, compared with BACE1+/+ mice. However, levels of full-length NCAM2 (NCAM2-TM and NCAM2-GPI) were similar in BACE1+/+ and BACE1−/− mice. E–G , representative immunoblot of membrane fractions of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using two different anti-C-terminal NCAM1 (rabbit pAb; AB5032 [ E ] and mouse mAb; 0B11 [ F ]) and anti-C-terminal NCAM2 (goat pAb; GTX89311 [ G ]) antibodies. After longer exposure, a ∼75 kDa NCAM1-CTF was detected in BACE1+/+ mice, but it was greatly decreased in BACE1−/− mice, and thus termed NCAM1-βCTF (arrow in D and E ). However, a BACE1-specific NCAM2-βCTF was not observed ( G ). H , sagittal section from 12-month-old BACE1+/+ olfactory bulb was stained with anti-BACE1 (3D5, magenta ), anti-NCAM2 (AF778, green ), and anti-NCAM1 (AB5032, red ) antibodies. NCAM1 and NCAM2 immunostaining with BACE1 are significantly overlapped in glomeruli. Scale bar represents 100 μm. D (BACE1+/+; n = 5–6, BACE1−/−; n = 5); E–G (BACE1+/+; n = 3 and BACE1−/−; n = 3); H (BACE1+/+; n = 4, two sagittal ( H ) and two coronal [ Fig. S3 ] sections).

Article Snippet: The membrane was then incubated overnight at 4 °C with the following primary antibodies. anti-NCAM2 (mouse mAb, 1:1000; sc-136328; Santa Cruz), anti-NCAM2 (goat pAb, 1:6000; GTX89311; GeneTex), anti-NCAM1 (goat pAb, 1:2000; AF2408; R&D systems), anti-NCAM1 (mouse mAb, 1:400; clone 5B8; DSHB), anti-NCAM1 (mouse mAb, 1:4000; clone 0B11; MilliporeSigma), anti-NCAM1 (rabbit pAb, 1:8000; AB5032; MilliporeSigma), anti-PSA-NCAM1 (mouse mAb; 1:2000;clone 2-2B; MilliporeSigma), anti-Myc (rabbit pAb, 1:1000; 2272; Cell Signaling), anti-turboGFP (mouse mAb, 1:2000; clone OTI2H8; ORIGENE), anti-SEZ6 (rat mAb, 1:250; clone 14E5; kindly provided by Dr S. Lichtenthaler) ( ) and anti-sAPPβ (mouse mAb, 1:40; clone BAWT kindly provided by Dr S. Lichtenthaler) , anti-β-tubulin (rabbit pAb, 1:1000; 2146; Cell Signaling), anti-BACE1 (rabbit mAb, 1:1500; D10E5; Cell signaling); anti-CHL1 (goat pAb, 1:2000; AF2147; R&D systems); anti-calnexin (mouse mAb, 1:1000; 610523; BD biosciences); anti-GAPDH (mouse mAb, 1:10,000; MAP374; Millipore), anti-PSD95 (rabbit pAb, 1:1500, ab18258, Abcam), and anti-synaptophysin 1 (mouse mAb, 1:1000, 101011; Synaptic System).

Techniques: Binding Assay, Western Blot, Staining, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: NCAM2 is cleaved by metalloproteinases or BACE1, followed by γ-secretase in HEK cells. HEK cells were transfected with NCAM2-Myc-DDK expression vector (transmembrane NCAM2 isoform) or empty vector (EV) as a control and then treated with DMSO only, GI254023X (GI; selective ADAM10 inhibitor, 5 μM in DMSO), C3 (β-secretase inhibitor IV, 10 μM in DMSO), GM6001 (GM; broad spectrum of MMPs inhibitor, 2.5 μM in DMSO), and DAPT (γ-secretase inhibitor, 10 μM, in DMSO) for 24 h as indicated. Total DNA concentration was kept constant at 1.5 μg with empty vector. A , representative immunoblot of cell lysates (Lysate) and conditioned media (CM) using anti-Myc (2272), anti-NCAM2 (sc-136328), anti-BACE1 (D10E5), and anti-GAPDH (MAB374) antibodies. Ectopically expressed full-length NCAM2 (NCAM2-FL) in HEK cells undergoes proteolysis with the generation of a C-terminal fragment (NCAM2-CTF) in cell lysates and secreted soluble fragments (sNCAM2) in conditioned media. GI and GM treatments produced a significant reduction of sNCAM2 levels indicating that NCAM2 is cleaved by ADAM10 and MMPs. DAPT treatment results in the accumulation of NCAM2-CTF in cell lysates indicating that NCAM2-CTF is further processed by γ-secretase. C , HEK cells were cotransfected with NCAM2 and BACE1 or EV as control and then treated with solvent only (DMSO), C3, or DAPT for 24 h. The ectopic expression of BACE1 produces BACE1-specific NCAM2-CTF (NCAM2-βCTF) and soluble NCAM2 fragment (sNCAM2β), and their production is completely inhibited by C3 treatment. Note that a different protein ladder was used in this figure compared to Figure 1 . B and D , graphs represent densitometric quantification of NCAM2-CTF/NCAM2-FL, NCAM2-FL/GAPDH, NCAM2-CTF/GAPDH ratio in lysates, and secreted soluble NCAM2 (sNCAM2 or sNCAM2β) in CM. One-way ANOVA with Tukey's multiple comparison test was applied. ∗ p < 0.05, ∗∗<0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, A and B , n = 3; C and D , n = 4. Full-length versions of the western blots in C are shown in Figure S4A . ns, not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C with the following primary antibodies. anti-NCAM2 (mouse mAb, 1:1000; sc-136328; Santa Cruz), anti-NCAM2 (goat pAb, 1:6000; GTX89311; GeneTex), anti-NCAM1 (goat pAb, 1:2000; AF2408; R&D systems), anti-NCAM1 (mouse mAb, 1:400; clone 5B8; DSHB), anti-NCAM1 (mouse mAb, 1:4000; clone 0B11; MilliporeSigma), anti-NCAM1 (rabbit pAb, 1:8000; AB5032; MilliporeSigma), anti-PSA-NCAM1 (mouse mAb; 1:2000;clone 2-2B; MilliporeSigma), anti-Myc (rabbit pAb, 1:1000; 2272; Cell Signaling), anti-turboGFP (mouse mAb, 1:2000; clone OTI2H8; ORIGENE), anti-SEZ6 (rat mAb, 1:250; clone 14E5; kindly provided by Dr S. Lichtenthaler) ( ) and anti-sAPPβ (mouse mAb, 1:40; clone BAWT kindly provided by Dr S. Lichtenthaler) , anti-β-tubulin (rabbit pAb, 1:1000; 2146; Cell Signaling), anti-BACE1 (rabbit mAb, 1:1500; D10E5; Cell signaling); anti-CHL1 (goat pAb, 1:2000; AF2147; R&D systems); anti-calnexin (mouse mAb, 1:1000; 610523; BD biosciences); anti-GAPDH (mouse mAb, 1:10,000; MAP374; Millipore), anti-PSD95 (rabbit pAb, 1:1500, ab18258, Abcam), and anti-synaptophysin 1 (mouse mAb, 1:1000, 101011; Synaptic System).

Techniques: Transfection, Expressing, Plasmid Preparation, Concentration Assay, Western Blot, Produced

Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: Identification and validation of the BACE1 cleavage site in NCAM2 and NCAM1. HEK cells were cotransfected with BACE1 and transmembrane NCAM2 (NCAM2-TM) or NCAM1-140. NCAM2-FL and NCAM2-βCTFs; or NCAM1-FL and NCAM1-βCTF were immunoprecipitated in cell lysates using anti-Myc (911B) antibody with agarose beads. After electrophoresis of immunoprecipitated samples, Coomassie stained bands of NCAM2-βCTF (∼32 kDa, in Fig. 2 C ) and NCAM1-βCTF (∼38 kDa, in Fig. 3 C ) were cut from the gel and sequenced by microcapillary LC/MS/MS to identify the BACE1 cleavage site. NCAM2-FL and NCAM1-FL were also sequenced as a control. A , comparison of BACE1 cleavage sites on mouse and human APP, NCAM2, and NCAM1. BACE1 cleaves APP at Asp1 within Aβ peptides ( yellow box ) and also cleaves APP at Glu11 (G 680 Y 681 ↓ E 682 V 683 ) within Aβ peptides, which are located 28 and 18 residues distant from the transmembrane region ( gray box ), respectively. APP numbering is based on the 770 amino acid isoform. NCAM2 and NCAM1 numbering is that of the mouse NCAM2-TM and mouse NCAM1-180 amino acid isoform, respectively. The ↓ symbol denotes the scissile (cleavage) bond. BACE1 cleavage sites of NCAM2 (G 661 Y 662 ↓ E 663 V 664 ) and NCAM1 (E 669 Y 670 ↓ E 671 V 672 ) in the second fibronectin type-III repeat domain ( green box , see FN2 in Fig. 1 C ) are located 35 and 41 residues distant from the transmembrane region ( gray box ), respectively. Notably, these cleavage sites are identical or similar to the secondary BACE1 cleavage site (β′) of human APP site (GY↓EV). The cleavage sites of NCAM2 at Asp 693 are indicated by the arrowhead. B and C , ion chromatogram of the peptide peak (EVQITAANR; m/z = 501.27–501.29) from the stained bands (32-kDa NCAM2-βCTF and NCAM2-FL as control). D and E , ion chromatogram of the peptide peak (EVQITAANR; m/z = 682.34–682.35) from the stained bands (38-kDa NCAM1-βCTF and NCAM1-FL as control). F and G , BACE1 cleavage site is denoted by a down arrow depicting the scissile bond (P1-P1′). All of the site-directed mutageneses in P1-P1′ amino acids are denoted. HEK cells were cotransfected with BACE1 and wild-type (WT) NCAM or mutant NCAM and then conditioned for 24 h with solvent only (DMSO) or BACE inhibitor (C3, 10 μM) dissolved in DMSO. Total DNA concentration was kept constant at 1.5 μg with empty vector. Cell lysates were analyzed by immunoblot to assess the BACE1 processing of mutant NCAM2 (E663H, YE662/663AH, E663K, YE662/663VK) ( F ) or NCAM1 (E671H, YE670/671AH, E671K, YE670/671VK) ( G ) compared with WT. When BACE1 is overexpressed, only NCAM2-YE662/663AH prohibits the generation of BACE1-specific 32-kDa NCAM2-βCTF ( arrowhead ). Instead, additional bands are produced, but these bands are abolished by BACE inhibition (C3). These data indicate that BACE1 cleaves NCAM2 between Y 662 and E 663 . NCAM1-E671H mutation did not prevent the generation of NCAM1-βCTFs at ∼38 kDa and ∼45 kDa ( arrowhead ). The ectopic expression of NCAM1-YE670/671AH, NCAM1-E671K, and NCAM1-YE670/671VK generates additional C-terminal fragments of NCAM1 at ∼41 kDa (41-kDa NCAM1-CTF) compared with NCAM1-WT even in the absence of BACE1 expression ( open arrowhead ). The coexpression of NCAM1-E671K with BACE1 resulted in increased 38-kDa NCAM1-βCTF levels, concurrently with reduced 45-kDa NCAM1-βCTF levels, most likely by favoring the proteolysis of NCAM1 at Glu 671 by BACE1. In contrast, only double mutations at the P1-P1′ site of NCAM1 (YE670/671AH and YE670/671VK) abolished the generation of BACE1-specfic 38-kDa NCAM1-βCTF and 45-kDa NCAM1-βCTF, but generated 41-kDa NCAM1-CTF, which was reduced by BACE inhibition (C3). These data support that BACE1 cleaves NCAM1 between Y 670 and E 671 . F and G , n = 3.

Article Snippet: The membrane was then incubated overnight at 4 °C with the following primary antibodies. anti-NCAM2 (mouse mAb, 1:1000; sc-136328; Santa Cruz), anti-NCAM2 (goat pAb, 1:6000; GTX89311; GeneTex), anti-NCAM1 (goat pAb, 1:2000; AF2408; R&D systems), anti-NCAM1 (mouse mAb, 1:400; clone 5B8; DSHB), anti-NCAM1 (mouse mAb, 1:4000; clone 0B11; MilliporeSigma), anti-NCAM1 (rabbit pAb, 1:8000; AB5032; MilliporeSigma), anti-PSA-NCAM1 (mouse mAb; 1:2000;clone 2-2B; MilliporeSigma), anti-Myc (rabbit pAb, 1:1000; 2272; Cell Signaling), anti-turboGFP (mouse mAb, 1:2000; clone OTI2H8; ORIGENE), anti-SEZ6 (rat mAb, 1:250; clone 14E5; kindly provided by Dr S. Lichtenthaler) ( ) and anti-sAPPβ (mouse mAb, 1:40; clone BAWT kindly provided by Dr S. Lichtenthaler) , anti-β-tubulin (rabbit pAb, 1:1000; 2146; Cell Signaling), anti-BACE1 (rabbit mAb, 1:1500; D10E5; Cell signaling); anti-CHL1 (goat pAb, 1:2000; AF2147; R&D systems); anti-calnexin (mouse mAb, 1:1000; 610523; BD biosciences); anti-GAPDH (mouse mAb, 1:10,000; MAP374; Millipore), anti-PSD95 (rabbit pAb, 1:1500, ab18258, Abcam), and anti-synaptophysin 1 (mouse mAb, 1:1000, 101011; Synaptic System).

Techniques: Immunoprecipitation, Electrophoresis, Staining, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Concentration Assay, Plasmid Preparation, Western Blot, Produced, Inhibition, Expressing, Generated

Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: Differential spatiotemporal BACE1 processing of NCAM1 and NCAM2 in vivo . A , representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fraction of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice. Well-characterized BACE1 substrates in vivo are also analyzed in the HC and OB at three different ages using anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), BACE1 (D10E5), anti-calnexin (610523), and anti-GAPDH (MAB374) antibodies. After increasing the contrast of the image for sNCAM1, sNCAM1β was detected in HC and OB of BACE1+/+ mice at P10. Transmembrane (TM) and GPI-anchored (GPI) NCAM2 isoforms were observed in the OB membrane fraction, while only NCAM2-TM was detected in HC membrane fraction. Full-length NCAM1 levels (180, 140, and 120) were observed in membrane fraction. Notably, full-length NCAM1 levels are significantly decreased at P10. B–I , graphs represent densitometry of soluble protein normalized to GAPDH or densitometry of membrane protein normalized to calnexin. White and gray bars represent BACE1+/+ and BACE1−/− mice, respectively. Unpaired two tailed t -test was used for analysis. ∗ p < 0.05, ∗∗<0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns, not significant, N.A.; not applicable, P10 (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5), 4 months (BACE1+/+; n = 5–6, BACE1−/−; n = 5), 12 months (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5). Full-length versions of the western blots (sNCAM2β and sNCAM1β) of 4-months-old mice shown in Figure S1, A and C .

Article Snippet: The membrane was then incubated overnight at 4 °C with the following primary antibodies. anti-NCAM2 (mouse mAb, 1:1000; sc-136328; Santa Cruz), anti-NCAM2 (goat pAb, 1:6000; GTX89311; GeneTex), anti-NCAM1 (goat pAb, 1:2000; AF2408; R&D systems), anti-NCAM1 (mouse mAb, 1:400; clone 5B8; DSHB), anti-NCAM1 (mouse mAb, 1:4000; clone 0B11; MilliporeSigma), anti-NCAM1 (rabbit pAb, 1:8000; AB5032; MilliporeSigma), anti-PSA-NCAM1 (mouse mAb; 1:2000;clone 2-2B; MilliporeSigma), anti-Myc (rabbit pAb, 1:1000; 2272; Cell Signaling), anti-turboGFP (mouse mAb, 1:2000; clone OTI2H8; ORIGENE), anti-SEZ6 (rat mAb, 1:250; clone 14E5; kindly provided by Dr S. Lichtenthaler) ( ) and anti-sAPPβ (mouse mAb, 1:40; clone BAWT kindly provided by Dr S. Lichtenthaler) , anti-β-tubulin (rabbit pAb, 1:1000; 2146; Cell Signaling), anti-BACE1 (rabbit mAb, 1:1500; D10E5; Cell signaling); anti-CHL1 (goat pAb, 1:2000; AF2147; R&D systems); anti-calnexin (mouse mAb, 1:1000; 610523; BD biosciences); anti-GAPDH (mouse mAb, 1:10,000; MAP374; Millipore), anti-PSD95 (rabbit pAb, 1:1500, ab18258, Abcam), and anti-synaptophysin 1 (mouse mAb, 1:1000, 101011; Synaptic System).

Techniques: In Vivo, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: BACE1 proteolysis of SEZ6, APP, and CHL1 in the hippocampus and olfactory bulb with age. A , representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fractions of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice. Well-characterized BACE1 substrates are also analyzed in the hippocampus and olfactory bulb at three different ages using anti-SEZ6 (14E5; kindly provided by Dr S. Lichtenthaler), anti-CHL1 (AF2147), anti-sAPPβ (BAWT; kindly provided by Dr S. Lichtenthaler), anti-APP (C1/6.1), anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), anti-BACE1 (D10E5), anti-GAPDH (MAB374), and anti-calnexin (610523) antibodies. The molecular weight of sSEZ6β in HC (∼155 kDa, arrowhead ) is different from the one in the OB (∼165 kDa, double arrowheads ) at the age of 4 and 12 months. The weak band at ∼140 kDa ( asterisk ) represents immature SEZ6-FL, while the strong band at 165-kDa ( open arrowhead ) or 175-kDa ( double open arrowheads ) in the OB represents mature SEZ6-FL. The different molecular weight of sSEZ6β or SEZ6-FL between the HC and OB of 4- and 12-month-old mice is most likely due to alternative splicing or posttranslational modifications. In the HC membrane fraction, CHL1-FL was detected at ∼185 kDa at P10, while CHL1-FL is detected as two bands at ∼185 kDa (plain arrow) and ∼175 kDa (dash arrow) in 4- and 12-month-old mice, most likely due to splicing variant or glycosylation. B–G , graphs represent densitometry of soluble protein (sSEZ6β, sAPPβ, and sCHL1) normalized to GAPDH or densitometry of membrane protein (mature SEZ6-FL, APP-FL, and 185-kDa CHL1) normalized to calnexin. White and gray bars represent BACE1+/+ and BACE1−/− mice, respectively. Unpaired two tailed t -test was used for analysis. ∗ p < 0.05, ∗∗<0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns; not significant, P10 (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5), 4 months (BACE1+/+; n = 5–6, BACE1−/−; n = 5), 12 months (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5).

Article Snippet: The membrane was then incubated overnight at 4 °C with the following primary antibodies. anti-NCAM2 (mouse mAb, 1:1000; sc-136328; Santa Cruz), anti-NCAM2 (goat pAb, 1:6000; GTX89311; GeneTex), anti-NCAM1 (goat pAb, 1:2000; AF2408; R&D systems), anti-NCAM1 (mouse mAb, 1:400; clone 5B8; DSHB), anti-NCAM1 (mouse mAb, 1:4000; clone 0B11; MilliporeSigma), anti-NCAM1 (rabbit pAb, 1:8000; AB5032; MilliporeSigma), anti-PSA-NCAM1 (mouse mAb; 1:2000;clone 2-2B; MilliporeSigma), anti-Myc (rabbit pAb, 1:1000; 2272; Cell Signaling), anti-turboGFP (mouse mAb, 1:2000; clone OTI2H8; ORIGENE), anti-SEZ6 (rat mAb, 1:250; clone 14E5; kindly provided by Dr S. Lichtenthaler) ( ) and anti-sAPPβ (mouse mAb, 1:40; clone BAWT kindly provided by Dr S. Lichtenthaler) , anti-β-tubulin (rabbit pAb, 1:1000; 2146; Cell Signaling), anti-BACE1 (rabbit mAb, 1:1500; D10E5; Cell signaling); anti-CHL1 (goat pAb, 1:2000; AF2147; R&D systems); anti-calnexin (mouse mAb, 1:1000; 610523; BD biosciences); anti-GAPDH (mouse mAb, 1:10,000; MAP374; Millipore), anti-PSD95 (rabbit pAb, 1:1500, ab18258, Abcam), and anti-synaptophysin 1 (mouse mAb, 1:1000, 101011; Synaptic System).

Techniques: Western Blot, Molecular Weight, Variant Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: NCAM1 but not NCAM2 is a BACE1 substrate in hippocampal synaptosomes of adult mice. Four-month-old BACE1+/+ and BACE1−/− hippocampi were used to isolate the synaptosome using discontinuous sucrose ultracentrifugation. A , representative immunoblot of purified hippocampal synaptosomes using anti-NCAM1 (AF2408), anti-NCAM1 (5B8), anti-NCAM2 (sc-136328), anti-SEZ6 (14E5), anti-CHL1 (AF2147), anti-APP (C1/6.1), anti-BACE1 (D10E5), anti-PSD95 (ab18258), anti-synaptophysin1 (101011), and anti-β-tubulin (2146) antibodies. Increased levels of NCAM1-FL (NCAM1-180 and NCAM1-140), CHL1-FL, APP-FL, and SEZ6-FL were detected in hippocampal synaptosomes. B , graphs represent densitometry of full-length NCAM1 (NCAM1-180, NCAM1-140, and NCAM1-120) protein with anti-NCAM1 (AF2408) or anti-NCAM1 (5B8) antibody normalized to β-tubulin in synaptosome. Unpaired two tailed t -test was used for analysis. ∗ p < 0.05, ∗∗ p < 0.001, BACE1+/+; n = 5, BACE1−/−; n = 5. N.A., not applicable; ns, not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C with the following primary antibodies. anti-NCAM2 (mouse mAb, 1:1000; sc-136328; Santa Cruz), anti-NCAM2 (goat pAb, 1:6000; GTX89311; GeneTex), anti-NCAM1 (goat pAb, 1:2000; AF2408; R&D systems), anti-NCAM1 (mouse mAb, 1:400; clone 5B8; DSHB), anti-NCAM1 (mouse mAb, 1:4000; clone 0B11; MilliporeSigma), anti-NCAM1 (rabbit pAb, 1:8000; AB5032; MilliporeSigma), anti-PSA-NCAM1 (mouse mAb; 1:2000;clone 2-2B; MilliporeSigma), anti-Myc (rabbit pAb, 1:1000; 2272; Cell Signaling), anti-turboGFP (mouse mAb, 1:2000; clone OTI2H8; ORIGENE), anti-SEZ6 (rat mAb, 1:250; clone 14E5; kindly provided by Dr S. Lichtenthaler) ( ) and anti-sAPPβ (mouse mAb, 1:40; clone BAWT kindly provided by Dr S. Lichtenthaler) , anti-β-tubulin (rabbit pAb, 1:1000; 2146; Cell Signaling), anti-BACE1 (rabbit mAb, 1:1500; D10E5; Cell signaling); anti-CHL1 (goat pAb, 1:2000; AF2147; R&D systems); anti-calnexin (mouse mAb, 1:1000; 610523; BD biosciences); anti-GAPDH (mouse mAb, 1:10,000; MAP374; Millipore), anti-PSD95 (rabbit pAb, 1:1500, ab18258, Abcam), and anti-synaptophysin 1 (mouse mAb, 1:1000, 101011; Synaptic System).

Techniques: Western Blot, Purification, Two Tailed Test