Mouse CHL-1/L1CAM-2 Antibody

Detection of Mouse CHL-1/L1CAM-2 by Western Blot

Axon guidance defects were absent in hippocampus mossy fibers of aged BACE1 cKO mice following partial BACE1 deletion. (a) Coronal sections collected from aged mice were stained with anti-synaptoporin (SPO) antibody (green) and DAPI (blue). Scale bar 50 μm. (b) Quantification of IPB length showed no alteration in TAM-treated mice compared to controls. IPB length was normalized on the length of the CA3 stratum lucidum (VEH n = 8; TAM n = 7, 3 to 4 sections per mouse). (c) Representative microscopy images showing reduced BACE1 (D10E5) expression in the hippocampus of TAM-treated mice. BACE1 signal was totally absent in BACE−/− mice, used as control to evaluate the amount of background in the staining. Scale bar 200 μm. Hippocampus full homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for analysis of APP processing and fractionated (soluble and membrane fractions) for the analysis of SEZ6 and CHL1 processing. Homogenates from aged-matched BACE+/− and BACE1−/− were loaded as control samples. Representative blots of (d) APP-FL (C1/6.1) and APP- CTFs (C1/6.1), (e) fractionation blots of sAPP beta (BAWT), SEZ6 (14E5) and CHL1 (AF2147). (f) Densitometry analysis of protein expression. APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the betaCTFs pC99 and pC89, and sAPP beta. betaCTFs and sAPP beta were identified because missing in the BACE1−/− sample. SEZ6 processing was decreased in TAM-treated mice with accumulation of the full length and decreased levels of the ectodomain (SEZ6-NTF) as well as decreased SEZ6-NTF/SEZ6FL ratio. Processing of CHL1 was also impaired as showed by increased of CHL1-FL levels, while CHL1-NTF was not altered. CHL1-NTF/CHL1-FL ratio was significantly decreased. APP-FL, CTFs, SEZ6-NTF and CHL1-NTF were normalized to GAPDH (MAB374), SEZ6-FL and CHL1-FL were normalized to Calnexin (610523) (VEH n = 5; TAM n = 5). (g) A betax-40 was quantified from hippocampus homogenates by MSD immunoassay. TAM-treated group displayed a significant reduction of A betax-40 levels (~50% decrease) compared to control (VEH n = 7; TAM n = 7). Results were plotted as Mean ± SEM, **p < 0.005; ***p < 0.001; n.s. = not significant, Student’s t test. DG: dentate gyrus, IPB: infrapyramidal bundle, slu: stratum lucidum, MB: main bundle. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31882662), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse CHL-1/L1CAM-2 by Western Blot

BACE1-mediated processing of APP and CHL1 is reduced in cortex of young BACE1 cKO mice following tamoxifen treatment. Cortex homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for Western blot analysis of APP and CHL1 processing. Homogenates from aged-matched BACE+/− and BACE1−/− were also loaded as control samples. Representative blots of (a) APP-full length (APP-FL) (C1/6.1), (b) APP-Carboxy Terminal Fragments (CTFs) (C1/6.1) and (c) CHL1. (d) Densitometry analysis of protein expression. Protein amount was normalized to protein levels in control mice (set at 1). APP-FL, pC99 and pC89 were normalized to GAPDH (MAB374) while CHL1-FL and CHL1-NTF were normalized to beta-tubulin (JDR.3B8). APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the betaCTFs pC99 and pC89. betaCTFs were clearly identified because missing in the BACE1−/− sample. CHL1-FL (AF2147) levels were increased while CHL1-N Terminal Fragment (CHL1-NTF) levels were not affected in cortex of TAM-treated mice. However, the CHL1-NTF/CHL1-FL ratio was significantly decreased in TAM-treated mice demonstrating reduced BACE1 processing (VEH n = 8; TAM n = 8). (e) A betax-40 was quantified from brain homogenates by ELISA (VEH n = 8; TAM n = 8). Levels of A betax-40 expressed as pMol/g of cortex were significantly reduced in TAM-treated mice (~50% decrease). Results were plotted as Mean ± SEM, ***p < 0.001; ****p < 0.0001; n.s. = not significant, Student’s t test. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31882662), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Mouse CHL-1/L1CAM-2 Antibody by Western Blot

Quantitative proteomics in Rab35 cKO P0 hippocampus.a Volcano plot of the TMT-based quantitative proteomes identifying the dysregulated proteins in Rab35 cKO hippocampus in comparison with the control hippocampus (n = 5 mice per genotype). b Number of proteins identified as significantly dysregulated and as either membrane traffic-related or neuronal migration-related. c, d Western blot analysis of control and Rab35 cKO P0 hippocampi using anti-contactin-2, anti-CHL1, and anti-actin antibodies. e, f Quantification of contactin-2 (c) and CHL1 (d) protein levels in control and Rab35 cKO P0 hippocampi. Band intensities of the indicated proteins were normalized to those of actin (n = 9 mice per genotype). Unpaired Student’s t-test; e, p = 0.0153; f, p = 0.0095. g, h Levels of contactin-2 (g) and CHL1 (h) were quantified by targeted MS using the PRM method (n = 5 mice per genotype). Unpaired Student’s t-test; gp = 0.0053; hp = 0.0229. i Western blot analysis of control and Rab35 cKO P0 hippocampus using anti-N-cadherin and anti-actin antibodies. j Quantification of N-cadherin protein levels in the control and Rab35 cKO P0 hippocampus (n = 9 mice per genotypes). Unpaired Student’s t-test, p = 0.9020. k Representative images of DIV 2 hippocampal primary neurons stained for contactin-2 (green), rhodamine-phalloidin (magenta) and DAPI (blue). Scale bar, 20 μm. l Quantification of contactin-2 intensity at the somatic plasma membrane in control (n = 4) and Rab35-deficient (n = 4) cells. Thirty neurons from four different cultures per genotype were analyzed. Mann–Whitney U-test, p = 0.0286. Data represent the mean ± SEM; n.s. not significant (p > 0.05); *p < 0.05; **p < 0.01. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/37085665), licensed under a CC-BY license. Not internally tested by R&D Systems.