44-854G

antibody from Invitrogen Antibodies

Targeting: CTTN EMS1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

44-854G

Provider

Invitrogen Antibodies

Product name

Phospho-Cortactin (Tyr421) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

References

Submitted references

Tumor necrosis factor α-mediated restructuring of the Sertoli cell barrier in vitro involves matrix metalloprotease 9 (MMP9), membrane-bound intercellular adhesion molecule-1 (ICAM-1) and the actin cytoskeleton.

Lydka M, Bilinska B, Cheng CY, Mruk DD
Spermatogenesis 2012 Oct 1;2(4):294-303

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Doubles game: Src-Stat3 versus p53-PTEN in cellular migration and invasion.

Mukhopadhyay UK, Mooney P, Jia L, Eves R, Raptis L, Mak AS
Molecular and cellular biology 2010 Nov;30(21):4980-95

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p53 suppresses Src-induced podosome and rosette formation and cellular invasiveness through the upregulation of caldesmon.

Mukhopadhyay UK, Eves R, Jia L, Mooney P, Mak AS
Molecular and cellular biology 2009 Jun;29(11):3088-98

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Paxillin-Y118 phosphorylation contributes to the control of Src-induced anchorage-independent growth by FAK and adhesion.

Sachdev S, Bu Y, Gelman IH
BMC cancer 2009 Jan 12;9:12

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Formation of extracellular matrix-digesting invadopodia by primary aortic smooth muscle cells.

Furmaniak-Kazmierczak E, Crawley SW, Carter RL, Maurice DH, Côté GP
Circulation research 2007 May 11;100(9):1328-36

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Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells.

Brábek J, Constancio SS, Siesser PF, Shin NY, Pozzi A, Hanks SK
Molecular cancer research : MCR 2005 Jun;3(6):307-15

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Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton.

Head JA, Jiang D, Li M, Zorn LJ, Schaefer EM, Parsons JT, Weed SA
Molecular biology of the cell 2003 Aug;14(8):3216-29

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on membrane extracts (30 µg lysate) of HeLa (Lane 1), and HeLa treated with Hydrogen peroxide (Lane 2). The blots were probed with Rabbit Anti-Phospho-Cortactin pTyr421 Polyclonal Antibody (Product # 44-854G, 1:250 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 62 kDa band corresponding to Phospho-Cortactin (Tyr421) was observed and was enhanced upon treatment in the given cell line. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-Cortactin (Tyr421) was performed using 70% confluent log phase NIH/3T3 cells treated with 1.6 mM hydrogen peroxide for 1 h. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-Cortactin (Tyr421) Rabbit Polyclonal Antibody (Product # 44-854G) at 1:250 dilution 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 represents the merged image showing cytoplasmic localization. Panel e shows the untreated cells. Panel f shows the control without primary antibody. The images were captured at 60X magnification.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3. Effects of TNFalpha on the steady-state levels of integral membrane, scaffolding and regulatory proteins in Sertoli cells in vitro. Sertoli cells were cultured at high density on Matrigel(tm)-coated 12-well dishes as described in Materials and Methods. On day 4 (designated as 0 h in this figure), TNFalpha (25 ng/ml) was added into Sertoli cell cultures, and cells were terminated at specific time points thereafter for lysate preparation. TNFalpha was dissolved in 10 mM NaH 2 PO 4 pH 7.4 at 22degC containing 0.15 M NaCl and 0.1% BSA (wt/vol). The control consisted of culturing Sertoli cells in media containing an equivalent amount of BSA. ( A ) Immunoblots of selected proteins involved in the regulation and in the maintenance of Sertoli cell barrier function. Actin served as an indicator of equal protein loading. ( B ) Histograms summarizing results shown in ( A ) from at least three independent experiments. Histograms are not shown for proteins whose levels did not change significantly. Each data point was normalized against its corresponding actin data point and then against the protein level at 0 h, which was arbitrarily set as 1. Each bar represents the mean +- SD of n = 3-4 experiments. **p < 0.01 (ANOVA followed by Dunnett&apos;s post-hoc test). n.d., not determined.