MA5-12394

antibody from Invitrogen Antibodies

Targeting: CD44 CD44R, CSPG8, HCELL, IN, MC56, MDU2, MDU3, MIC4, Pgp1

Provider product page for MA5-12394

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [24]
Comments [0]
Validations
Immunocytochemistry [1]
Other assay [7]

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Product number: MA5-12394 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD44 Monoclonal Antibody (5F12)
Antibody type: Monoclonal
Antigen: Other
Description: MA5-12394 has been successfully used in immunofluorescence analysis of CD44 in human bone marrow derived mesenchymal stem cells.
Antibody clone number: 5F12
Concentration: 0.2 mg/mL

CD44 protein structure - MA5-12394 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 SRGN/CD44 axis induces CLDN1 expression via NF-kappaB activation. ( a ) NF-kappaB reporter assay was performed in H1299/Mock and H1299/SRGN cells. ( b ) Cells were cultured in serum-free medium for 48 h, and subjected to western blot analysis using anti-IkappaBalpha (#4812, Cell Signaling Technology) and anti-p65 (sc-372, Santa Cruz Biotech). ( c ) H1299/SRGN cells were transiently transfected with vectors encoding dominant-negative IkappaB kinase alpha (DN-IKKalpha) or DN-IKKbeta and cultured in serum-free medium for 48 h, followed by western blot analysis of IkappaB and CLDN1. HA-fused DN-IKKs were detected by anti-hemagglutinin (sc-805, Santa Cruz Biotech). ( d ) Unsorted H1299 and CD44(-) cells stably harboring the Mock-control or SRGN-expressing vectors were cultured in serum-free medium for 48 h. Nuclear and cytosolic fractions were prepared for western blot analysis using anti-p65, anti-PARP (sc-7150, Santa Cruz Biotech) and anti-tubulin (GTX112141, GeneTex). ( e ) Cells described in d were cultured in serum-free medium for 48 h, and subjected to western blotting of CD44 and CLDN1. ( f ) H1299 CD44(-) cells stably harboring Mock-control or CD44s-expressing vectors were incubated in medium supplemented with CM collected from H1299/Mock or H1299/SRGN cells for 24 h, and subjected to western blot analysis of CLDN1. Data are presented as the mean+-s.d. of three independent experiments. ** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 CD44 is critical for SRGN-instigated malignant phenotypes. ( a ) CD44-negative H1299 cells were enriched by three rounds of FACS procedure. ( b and c ) Western blot analysis was performed to show the expression of CD44 (using Hermes-3, ATCC) and SRGN (using anti-Flag M2, Sigma-Aldrich) in unsorted and CD44(-) cells ( b ), and in cells stably harboring the Mock-control and SRGN-expressing vectors ( c ). ( d ) Migration and invasion assay. ( e ) 2 x 10 6 cells were injected to NOD-SCID mice through tail-vein, and tumors developed in the liver (left panel) and lung (right panel) were assessed. ( f ) H1299 CD44(-) cells stably harboring Mock-control vector as well as vector encoding CD44s were subjected to Boyden chamber migration assay in the presence of CM collected from H1299/Mock and H1299/SRGN cells. Western blot analysis shows CD44 expression in H1299 CD44(-)/Mock and H1299 CD44(-)/CD44s cells. ( g , h ) Migration assays of HTB-40/Mock and HTB-40/CD44 cells incubated with SRGN-containing CM ( g ) or with serum-free medium supplemented with or without recombinant human SRGN (5 mug/ml) ( h ). Western blot analysis shows CD44 expression. Data are presented as the mean+-s.d. of three independent experiments. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3(A-E) The IGFBP-3 protein and WT peptide, but not the mutant, block HA-CD44 signaling and more effectively inhibit cell viability of A549 cells than either the p53-negative H1299 or CD44-negative HFL1 cell lines. IGFBP-3 protein or peptides were added to cells in the absence or presence of the CD44 antibody, 5F12, known to block HA-CD44 interactions, or 4-MU. Cell viability was assessed by the MTT assay. Cells were seeded in 96-well plates at 0.2 x 10 5 cells per well in 10% FBS-supplemented media. The following day, the cell monolayers were incubated in serum-free medium for 12h, then treated as indicated for 48h with the media containing the specific components in the different treatments replaced every 12h. The concentration of IGFBP-3 protein or peptides added was 50 nM and that of 4-MU was 600 muM. The CD44 antibody (5 mug/mL) was added either separately or 2h prior to addition of IGFBP-3 and/or peptides. Optical density measurements (570 nm) were normalized by expressing each point in relation to the untreated control of each cell line (set to 100%). Each column represents the mean +- S.D. of three independent experiments, each run in triplicate. Asterisks (*) indicate a statistically significant difference from the corresponding untreated cell line control, *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3(F-J) The IGFBP-5 and IGFBP-6 peptides are able to block HA-CD44 signaling and cell viability of A549 cells, albeit less effectively than the IGFBP-3 peptide. Cell viability was measured as described in ( A-E ) legend.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Both AChE concentration and activity are increased to a comparable extent in the media of A549 cells upon treatment with the IGFBP-3 protein or peptide, 5F12, 4-MU, or in combination. Cells (0.2 x 10 5 ) were grown in 10% FBS-supplemented media overnight then in serum-free medium for 12 h prior to treatment with mIgG (5 mug/mL), 5F12 (5 mug/mL), 50 nM IGFBP-3 protein or peptides, 600 uM 4-MU, or in combination. The cells were then incubated for 48 h and the media collected. Samples (3 uL of 600 ug/mL total protein) were spotted onto a nitrocellulose membrane and AChE was visualized using anti-AChE antibodies (Methods). The dots were quantitated, averaged, normalized and expressed as fold change relative to untreated cells ( A,C,E ). AChE activity using the same amount of protein was measured as described in Methods ( B,D,F ). The graphs prepared using the GraphPad 8.3.1 software, summarize the results expressed as means +- S.D. of five independent experiments, each performed in triplicate. Asterisks (*) indicate a statistically significant difference from the corresponding untreated cell line control, *p < 0.05, **p < 0.0 l of each cell line. Absence of asterisks indicates no significance, Mann-Whitney test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 WT IGFBP-3 l -peptide is more effective than the d -peptide in both binding HA and in blocking viability of A549 cells that express CD44 as compared to the CD44-negative cell line, HFL1. (A) IGFBP-3 peptides (50 n m each) were bound to the ELISA plate wells, and then, 200 n m biotin-HA was added and processed as described in the Materials and methods section. The data were normalized to the control incubated with BSA (control 1), and fold change relative to the control was calculated. Control 2 is a negative control that included bound WT IGFBP-3 l -peptide and added streptavidin-HRP and TMB without addition of biotin-HA. Each column represents the mean +- SD of three independent experiments, each run in triplicate. The asterisks (** P < 0.01) indicate a statistically significant difference from control 1 and of the IGFBP-3 l -peptide compared to the d -counterpart. The absence of asterisks indicates no significance, Mann-Whitney test. (B, C) IGFBP-3 peptides were added to cells in the absence or presence of the mIgG (5 mug*mL -1 ) antibody control or the CD44, 5F12 antibody (5 mug*mL -1 ), known to block HA-CD44 interactions. Cell viability was assessed by the MTT assay. Cells were seeded in 96-well plates at 0.2 x 10 5 cells per well in 10% FBS-supplemented media. The following day, the cell monolayers were incubated in serum-free medium for 12 h and then treated as indicated for 48 h with the media containing the specific components in the different treatments repla

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Treatment of cells with either 4-MU or 5F12 resulted in increased Abeta in the media of A549 and H1299 cells. Cells (0.2 x 10 5 ) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h, then treated as indicated for 72 h with 600 uM 4-MU and/or the CD44 antibody (5F12, 5 mug/mL). The media was collected then the same amount of protein (3 uL of 600 ug/mL total protein) of each sample not-treated (NT) or treated as indicated, was spotted onto a nitrocellulose membrane. The blots were stained with Ponceau ( A ). ( B ) Blots were incubated with anti-Abeta (6E10) antibodies, and the signal on the membrane was detected using super signal west pico luminol (chemiluminescence) reagent. The membranes were imaged with a Bio-Rad molecular imager, and quantitated with Image J. The dots from five independent assays, each carried out in triplicate, were quantitated, averaged, normalized, and expressed as fold change relative to untreated control cells after subtraction of the values for the blank consisting of dot blots probed with mouse IgG isotype control with no relevant specificity to a target antigen (mIgG, 5 mug/mL) or anti-Abeta (6E10) antibodies incubated with blots dotted with media not incubated with cells ( C ) using the GraphPad 8.4.3 software. The graphs summarize the results expressed as means +- SD (n = 5). Asterisks (*) indicate a statistically significant difference from the correspondi