PA3-16744

antibody from Invitrogen Antibodies

Targeting: CADM1 BL2, IGSF4, IGSF4A, Necl-2, NECL2, RA175, ST17, SYNCAM, SYNCAM1, TSLC1

Provider product page for PA3-16744

Western blot

Other assay

Antibody data

Antibody Data
Antigen structure
References [2]
Comments [0]
Validations
Western blot [5]
Other assay [4]

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Product number: PA3-16744 - Provider product page
Provider: Invitrogen Antibodies
Product name: CADM1 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: Suggested positive control: rat brain.
Reactivity: Human, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

CADM1 protein structure - PA3-16744 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: cells were transfected with the pCMV6-ENTRY control or pCMV6-ENTRY CADM1 cDNA for 48 hrs and lysed. Equivalent amounts of cell lysates (5 µg per lane) were separated by SDS-PAGE and immunoblotted with anti-CADM1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of CADM1 in rat brain and HEK cell lysates. Samples were incubated in CADM1 polyclonal antibody (Product # PA3-16744). Showing different glycosylation patterns. Brain fractions give banding patterns of a core glycosylation population around 50 kDa and a slightly higher glycosylated population around 65 kDa. The heterologous HEK cell lysates give a single, fully glycosylated, band around 107 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of CADM1 in 5 µg /lane cells transfected with the pCMV6-ENTRY control or pCMV6-ENTRY CADM1 cDNA. Samples were incubated in CADM1 polyclonal antibody (Product # PA3-16744). Cells were transfected with the pCMV6-ENTRY control or pCMV6-ENTRY CADM1 cDNA for 48 hrs and lysed. Equivalent amounts of cell lysates were separated by SDS-PAGE and immunoblotted.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of CADM1 in 0.5 mg/mL Human Brain lysate. Samples were incubated in CADM1 polyclonal antibody (Product # PA3-16744). This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-CADM1 Polyclonal Antibody (Product # PA3-16744) and a 100kDa band corresponding to Cell adhesion molecule 1 was observed across cell lines and tissue extract tested except K-562, Jurkat and A-431 which is reported to be low. Membrane enriched extracts (30 µg lysate) of Caco-2 (Lane 1), SK-O-V3 (Lane 2), Hep G2 (Lane 3), K-562 (Lane 4), Jurkat (Lane 5), A-431 (Lane 6) and tissue extract of Rat Lung (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 SynCAM1-3 function redundantly to set synapse density and size. (A) Representative 60x confocal microscopy image of cultured rat hippocampal neurons at 14 DIV treated with sub-saturating lentivirus carrying Scrambled1-3 amiRNAs linked to nlsGFP, and very low titer memGFP lentivirus. An individual pyramidal cell co-labeled by memGFP and nlsGFP is marked by a red arrowhead. White arrowheads mark nuclei in the field of view only expressing nlsGFP. Scale, 50 mum. (B) Sample images of automated puncta and synapse detection from co-immunostaining for presynaptic protein Synapsin 1 and postsynaptic protein PSD-95 following manual selection of an individual dendrite segment from the image in (A) . Synapses are defined as the co-localized regions of pre- and postsynaptic puncta (white areas in merged image). Scale, 10 mum. (C) Average synapse density and (D) average synapse puncta area from 13 to 15 DIV neurons co-transduced with memGFP and sub-saturating amounts of the SynCAM amiRNAs listed or control Scrambled1-3 amiRNAs. n = cells/isolations is listed on the bars. SKD, single knockdown; DKD, double knockdown; TKD, triple knockdown. * p < 0.05, ** p < 0.01, one-way analyses of variance (ANOVA) with Tukey's post hoc pairwise comparisons on cell average values. Error bars, SEM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 MEDLR shows synaptic phenotypes are due to postsynaptic SynCAM1-3 depletion. (A) Representative images of memGFP-labled basal dendrites from amiRNA- and amiRNA+ neurons at 15 DIV in cultures transduced with sub-saturating amounts of Scrambled1-3 or SynCAM1-3 amiRNAs. Automated puncta detection for PSD-95 and Synapsin 1 immunostaining and synapses (merge) was performed after manual selection of GFP masks. Scale, 5 mum. (B,D,F) Average dendritic puncta density as indicated on graph of amiRNA- and amiRNA+ neurons and (C,E,G) corresponding cumulative puncta density distribution plot (%) of amiRNA+ neurons from cultures transduced with Scrambled1-3 or SynCAM1-3 amiRNAs. (H,J,L) Average puncta area as indicated on graph of amiRNA- and amiRNA+ neurons and (I,K,M) corresponding cumulative puncta area distribution plot (%) of amiRNA+ neurons from cultures transduced with Scrambled1-3 or SynCAM1-3 amiRNAs. n = number of cells/isolations as indicated on bars from 13 to 15 DIV cultures. * p < 0.05, ** p < 0.01, *** p < 0.001, t test on cell average values. Cumulative distribution plots represent cell average values. Error bars, SEM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 MPP2 binds directly to the synaptic adhesion molecule SynCAM1. ( a ) Targeted identification of putative MPP2 PDZ domain interactors: The 10 C-terminal amino acids of known synaptic PDZ-ligand proteins (see table on the left) were fused to the monomeric mCherry tag and tested for interaction with MYC-tagged MPP2 in coimmunoprecipitation experiments. Pulldown control (MYC-MPP2) and coprecipitated proteins (mCherry-PDZ-Ligand) were detected by western blot (upper panels); input controls are shown below. For uncropped blots, see Supplementary Fig. S3 . ( b ) Isothermal titration calorimetry measurements with 350 muM mCherry-SynCAM1 and SynCAM1mut, respectively, injected to 55 muM MPP2-PSG module revealed a robust binding of the SynCAM1 C-terminus with a K d of 3.1 +- 0.239 muM (left panels). Results for the mutated SynCAM1 C-terminus control are shown on the right. ( c ) Full-length HA-tagged SynCAM1 (HA-SynCAM1) and MYC-tagged MPP2 (MYC-MPP2), HA-tagged mutant SynCAM1 (HA-SynCAM1mut) and MYC-MPP2, or HA-SynCAM1 and MYC-tagged MPP2 SH3-GK (MYC-MPP2-SH3GK) were coexpressed in COS7 cells and immunoprecipitated with alphaHA (mHA IP) or mouse IgGs (mIgG IP) as a negative control. Pulldown controls are shown (HA-SynCAM); coprecipitated proteins are detected by western blot with alphaMYC antibody below. For uncropped blots, see Supplementary Fig. S3 . ( d ) Immunoprecipitation of SynCAM1 (using a chicken SynCAM1-Biotin antibody or chicken IgYs as a negative control) from a cr

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Sub-saturating lentiviral artificial microRNA (amiRNA) transduction potently knocks down synaptic cell adhesion molecules 1-3 (SynCAM1-3) in amiRNA+ cells while keeping a subpopulation of wild type (WT) amiRNA- cells. (A) Lentiviral knockdown (KD) vectors were generated with a minimal CMV (mCMV) promoter, intronic amiRNAs, and nlsGFP. RSV, Rous sarcoma virus promoter; LTR, long-terminal repeat; RRE, Rev-response element; cPPT, central polypurine tract; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; SA, splice acceptor; SD, splice donor. (B) memGFP-expressing lentiviral vector for dendrite labeling. (C) Comparison of cadm1-3 mRNA levels at 14-15 days in vitro (DIV) by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in cultured rat hippocampal neurons using saturating viral titers relative to levels in uninfected control cultures. n = 2 experiments. (D) Transduction rate of cultured neurons at 14-15 DIV using sub-saturating viral titers. n = 2 experiments. (E) Representative blots and (F) relative SynCAM1-3 protein levels at 13-15 DIV measured by quantitative western blotting of cultured neurons following indicated amiRNA lentiviral treatments at saturating or sub-saturating viral titers compared to Scrambled1-3 amiRNA control treatments. Actin was used as a loading control. n = 4 (saturating) or n = 2 (sub-saturating) experiments. A single gel image of the same intensity was cropped as marked by dotted lines and rearrang