44-650G

antibody from Invitrogen Antibodies

Targeting: PTK2 FADK, FAK, FAK1, PPP1R71

Provider product page for 44-650G

Western blot

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [19]
Comments [0]
Validations
Flow cytometry [1]
Other assay [18]

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Product number: 44-650G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-FAK (Tyr407) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat, Chicken/Avian
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

PTK2 protein structure - 44-650G shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Combined proteomic and phosphoproteomic analysis of isolated adhesion complexes. ( a ) Schematic workflow for the isolation and proteomic/phosphoproteomic analysis of adhesion complexes. Cells were allowed to spread on FN or, as a control, Tf and complexes were isolated by a combination of crosslinking, cell lysis and a high-pressure wash to remove cell bodies. Collected complexes were analysed using either a proteomic or phosphoproteomic workflow, after which the FN-specific proteins and phosphoproteins were identified by performing a subtractive comparison with controls. ( b ) Immunoblot analysis of complexes isolated from cells spread on FN and Tf, as well as the WCLs of cells spread on FN. M, MW markers (kDa; values displayed to the left of each blot). Dashed lines indicate where images have been cropped for display purposes. ( c ) A Venn diagram showing the overlap between the FN-specific proteins (left circle) and phosphoproteins (right circle) identified by proteomic and phosphoproteomic analyses of isolated complexes, respectively. In addition to the total number of proteins (black text), the number of adhesome proteins identified in each data set is also displayed (red text). To the right of the panel, all 19 adhesome components identified exclusively by the phosphoproteomic analysis are displayed. Proteins in bold text were not identified by any other proteomic analyses of isolated FN-induced adhesion complexes.

Supportive validation

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Experimental details: FIGURE 6: VEGF binding to NRP1 is important for FAK Tyr-407 phosphorylation in endothelial cells. (A) HUVECs were infected with GFP, WT NRP1, or Y297A NRP1. Growth medium was replaced by serum-free medium 72 h after infection, and the following day quiescent infected cells were treated with VEGF-A 165 for the times indicated. Whole-cell extracts were then analyzed by using Western blotting with the indicated antibodies. Blots from panel A (n = 4) were quantified by scanning densitometry to determine the extent of phosphorylation KDR Y1175 and FAK Y407 (right). The bar graph shows the mean ratio of phosphorylated to total protein from four independent experiments +- SD; *p < 0.05 vs. WT 30 min VEGF treatment. (B) HUVECs were transfected with negative control siRNA (NC) or siRNAs targeted to NRP1 or NRP2, and 48 h later were treated with 25 ng/ml VEGF-A 165 for 10 or 30 min. Cells were then lysed and immunoblotted as indicated. Blots were quantified as described earlier; *p < 0.05 vs. control siRNA.

Supportive validation

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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's post-hoc test). n.d., not determined.

Supportive validation

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Experimental details: Figure 2. The cytosolic building blocks of cell-matrix adhesion sites are combinatorially diversified. ( A ) Not all cytosolic integrin-adhesome complexes are necessarily building blocks for adhesion sites. If each protein is in only one type of building blocks then physically associated proteins should exhibit the same dwell times ( tau 1/2 ) and mobile fractions in focal adhesions. ( B ) REF52 cells expressing the analyzed proteins tagged with meGFP were measured using FRAP and FCS to quantify their tau 1/2 and mobile fractions in focal adhesions and their dwell times in a confocal volume in the cytosol ( tau D ). ( C ) Example FRAP images before (0'') and after bleaching a focal adhesion (arrows). Scale bar, 5 mum. ( D - F ) Box plots of the tau D (26 >= n >= 14 cells), tau 1/2 and mobile fractions (31 >= n >= 7 cells) of each protein. ( G ) Median tau 1/2 vs median mobile fraction of each protein normalized to zero-mean and unit-variance. Thus, in this plot the Euclidean distance (co-dynamics distance) between proteins quantifies the difference in their dynamics. ( H ) The co-dynamics distance vs median association score of all possible 78 heteromeric protein pairs. ( I ) The reported vertical distance from substrate of 6 of the analyzed components across focal adhesions ( Kanchanawong et al., 2010 ) and the cytosolic associations between them as measured here ( Figure 1E ). Anchor, spring, and actin symbols indicate vertical layers of integrin signalling, mecha

Supportive validation

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Experimental details: Figure 3. Symmetric material exchange between stationary focal adhesions and cytosol. ( A ) The symmetric and asymmetric models of material exchange between adhesion sites and cytosol. In symmetric exchange a component, A , exits from adhesion site in the same state it had upon entering to it. In asymmetric exchange A exits in a different, primed state A* and relaxes back to state A in the cytosol, thereby generating a spatial gradient of the primed state emanating from adhesion sites. ( B ) Formulation of the two models for the case in which priming (of A ) is based on interaction (with protein B ). Here, asymmetric exchange would generate a spatial gradient of AB complex concentration emanating from adhesion sites. ( C - F ) Discriminating between the two models by measuring the physical associations near (= 9) ( E ) or for the median score of each pair +- MAD ( F ). Data-points far from the equality diagonals (dashed red lines) would correspond to asymmetric exchanges, as illustrated in ( D ), while data-points along the diagonal indicate symmetric exchange. Histogram in ( D ) shows the distribution of the difference in association scores near and far from focal adhesions. ( G ) FLIM images color-coding the fraction, alpha, of

Supportive validation

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Experimental details: Figure 3--figure supplement 1. FAK, paxillin, and CAS are tyrosine-phosphorylated and interact with SH2 domain in focal adhesions. ( A ) Immunofluorescence images of REF52 stained for FAK pY407, paxillin pY118, and CAS pY165 using phospho-specific antibodies. Scale bars, 15 mum. ( B ) A REF52 cell co-expressing meGFP-dSH2 and TD-mKate2-paxillin. Note the localization of dSH2 in cell-matrix adhesion sites, as previously reported ( Kirchner et al., 2003 ). Scale bar, 10 mum. ( C ) Top and middle rows, FLIM images color-coding the fraction (alpha) of the donor- (mCitrine) tagged protein (FAK, paxillin, CAS, or ILK) that FRET to the acceptor- (mCherry) tagged dSH2 before and after acceptor photobleaching. ILK is used here as a negative control for tyrosine phosphorylation. Note that low levels of alpha are observed also for ILK in focal adhesions, plausibly due to density effects. However, FAK, paxillin, and CAS exhibit higher alpha values, suggesting FRET due to direct interaction. Scale bars, 10 mum. DOI: http://dx.doi.org/

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Validation of adhesion complex-localized phosphorylation sites. ( a ) Immunoblot analyses of the protein complexes isolated from cells spread on FN or Tf for 120 min. Samples were probed using phosphospecific antibodies targeting: paxillin pTyr118; FAK pTyr397, pTyr576 and pTyr407; MYPT1 pThr696; cortactin pTyr421; SHP2 pTyr580; and PKCdelta pTyr313. ( b ) Immunofluorescence images of cells spread on FN for 120 min. Cells were stained using the phosphospecific antibodies FAK pTyr407, cortactin pTyr421 and PKCdelta pTyr313 (all green). In each case, cells were also stained using an antibody targeting the focal adhesion marker paxillin (red). Scale bar, 20 mum. ( c ) Immunoblot analyses of the WCLs of cells spread on FN or Tf for 120 min. Samples were probed using the same antibodies as used to probe isolated complexes in a . Dashed lines in a , c indicate where the images have been cropped for display purposes. White arrows to left of blots highlight the expected MWs of the corresponding proteins. Black arrows to the left of blots highlight a putative high MW isoform of PKCdelta.