- PA1-1045 - Invitrogen Antibodies NAMPT antibody

Submitted references eNAMPT Is Localised to Areas of Cartilage Damage in Patients with Hip Osteoarthritis and Promotes Cartilage Catabolism and Inflammation.

Philp AM, Butterworth S, Davis ET, Jones SW
International journal of molecular sciences 2021 Jun 23;22(13)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot detection of Visfatin from human adipose tissue lysate using Product # PA1-1045.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (20 µg lysate) of U-87 MG (Lane 1), PC-3 (Lane 2), A-431 (Lane 3), HCT 116 (lane 4) and Jurkat (lane 5). The blots were probed with Anti-Visfatin Rabbit Polyclonal Antibody (Product # PA1-1045, 1:250-1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 55 kDa band corresponding to Visfatin was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Visfatin was done on 70% confluent log phase U87MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Visfatin Rabbit Polyclonal Antibody (Product # PA1-1045) at 1 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Visfatin was done on U-87 MG cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Visfatin Rabbit Polyclonal Antibody (PA11045, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 The expression of visfatin in the serum and joint tissues of patients with hip OA. ( A ) Serum extracellular visfatin concentrations were determined by ELISA in n = 76 hip OA patients of varying BMI. ( B ) Expression of total visfatin under reducing conditions in w/w matched tissue samples (serum (S), cartilage (c), bone (B), synovium (Sy), synovial fluid (Sy.f), infrapatellar fat pad (IFP), adipose (A), and muscle (M)) ( n = 3 in total, representative blots shown). Recombinant visfatin (r.Vis) indicates the molecular weight region. ( C ) Confirmation of detection of visfatin in cartilage. Cartilage visfatin expression was confirmed through increasing cartilage (C) protein load and reducing adipose (A) and serum (S) protein load. ( D ) Tissue panel of a Western blot sample from normal-weight (NW) and obese (OB) individuals with hip OA. All samples were normalised to mug of total protein loaded, and an equal loading was confirmed by ponceau-S staining and actin expression. ( E ) Western blots were analysed using Image J software and the densitometry was compared in NW and OB patients. Relative density was calculated according to the protein loading control and plotted against patient BMI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Visfatin induces the production of cartilage catabolic proteases in hip OA cartilage and is co-localised with MMP13 at sites of damage. MMP secretion from cartilage explants following visfatin stimulation (500 ng/mL) and IL-1ss (1 ng/muL) using Luminex, and separated into the MMP classification ( n = 9 individual patients (five explants per patient)). ( A ) Collagenases classification. ( B ) Gelatinases classification. ( C ) Stromelysins classification. ( D ) Matrilysins and Metalloelastase classification. * = p < 0.05, ** = p < 0.01, significant difference between the treatment and control values. ( E ) IHC of cartilage on a human femoral head showing full-thickness and fibrillated cartilage: (i) H&E staining of full-thickness cartilage; (ii) fluorescent images of full-thickness cartilage (anti-visfatin shown in red, anti-NFkB shown in green; n = 4 individual patients)--solid arrow represents area of smooth cartilage, and the dotted arrow demonstrates low visfatin expression within the pericellular area of the chondrocytes; (iii) H&E staining of degraded and fibrillated cartilage (25x magnification); (iv) fluorescent images of degraded and fibrillated cartilage (anti-visfatin shown in red, anti-NFkB shown in green; n = 4 individual patients) (25x magnification)--solid arrow represents area of smooth cartilage, and the dotted arrow demonstrates increased visfatin expression within the pericellular area of chondrocytes. ( F ) Co-expression of MMP-13 and visfatin in de

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