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anti-phosphotyrosine

By Leslie Perkins,2014-06-11 23:49
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anti-phosphotyrosine

Detailed Legend for Each Figure:

    2+Fig.1. Preferential binding of paxillin to the 4-tail. a. Ni-charged resin was loaded with ?4, ?1A, ?4?1A, or ?IIb tail model proteins. Bound proteins from Jurkat T

    cell lysate (Ly) were separated on 4 - 20% SDS-PAGE under reducing conditions,

    transferred to Immobilon P membranes, and immunoblotted with antibodies specific for

    paxillin (clone 349), filamin (MAB1680, Chemicon), or talin (clone 8d4) and quantified

    by scanning densitometry. Coomassie blue staining of gels (Affinity matrix) assessed

    loading of each tail. Western blotting with anti-phosphotyrosine antibodies PY20 and

    4G10 did not reveal phosphotyrosine in the fraction of paxillin that bound to ?4 tail. b.

    2+Ni-charged resin was loaded with ?IIb, ?3A, ?4, ?5, or ?6A tail. Bound proteins and Jurkat T cell lysate (Ly) were separated on 4 - 20% SDS-PAGE, transferred to

    Immobilon P membranes, and immunoblotted with antibody specific for paxillin (clone

    349). Coomassie blue staining of gels (Affinity matrix) assessed loading of each model

    2+protein. c. Ni-charged resin was loaded with ?4, ?1A, ?4?1A, or ?IIb tail. Bound proteins from platelet lysate (Ly) were separated on 4 - 20% SDS-PAGE under reducing

    conditions, transferred to Immobilon P membranes, and immunoblotted with anti-paxillin

    (clone 349), which recognizes both paxillin and Hic-5

    92+. Ni-charged resin was loaded

    with ?4, ?1A, or ?IIb tail. Bound proteins from Jurkat T cell lysate (Ly) were separated

    on 4 - 20% SDS-PAGE under reducing conditions, transferred to Immobilon P

    membranes, and immunoblotted with anti-leupaxin antibody (rabbit polyclonal antibody

    raised against the N-terminal 14 amino acids (EELDALLEELERST) of human leupaxin).

    d. Recombinant paxillin, at the indicated concentration, was added to ?4 or ?IIb model

protein-loaded resins. Bound paxillin was separated by SDS-PAGE under reducing

    conditions, transferred to Immobilon P membranes, and detected with anti-HA tag

     of paxillin binding to ?4-50antibody. The quantity of bound paxillin was estimated by scanning densitometry using

    tail. All concentrations of bound paxillin were assayed within the linear range of the the NIH “Image” program and used for determination of EC

    standard curve.

    Fig.2. Association of paxillin with 41. a. Upper panel. Cell lysate from Jurkat T cells was precipitated with antibodies specific for human ?4 (HP2/1), ?1A (B-D15,

    BioSource), ?5 (PharMingen), or an irrelevant mouse IgG (IgG). Immunoprecipitated

    proteins were separated on 4 - 20% SDS-PAGE under reducing conditions, transferred to

    Immobilon P membranes, probed with biotin-labeled anti-paxillin antibody, and detected

    with streptavidin followed by ECL. Lower panel. In parallel, cell lysate from cell surface

    biotin-labeled Jurkat T cells were precipitated with same antibodies noted above.

    Immunoprecipitated proteins were separated on 6% SDS-PAGE under non-reducing

    conditions, transferred to Immobilon P membranes, and detected with streptavidin

    peroxidase followed by ECL. Positions of ?1, ?4 (150, 80, 70 kDa forms), ?5and

    ?1integrin subunits are indicated. b. Jurkat T cells were surface biotin-labeled. Aliquots of cell lysate were subjected to multiple rounds of immunoprecipitations using anti-

    paxillin antibody (paxillin-depleted) or an irrelevant IgG (mock-depleted). Cell lysates

    were then immunoprecipitated with either anti-?4 or anti-?5 antibody.

    Immunoprecipitated proteins were separated on 6% SDS-PAGE under non-reducing

    conditions, transferred to Immobilon P membranes, and detected with streptavidin

peroxidase followed by ECL (upper panel). Bottom panel: paxillin-depleted or mock-

    depleted cell lysates were separated on 4-20% SDS-PAGE and the quantity of remaining

    paxillin was determined by Western blot analysis. c. Jurkat T cells were surface biotin-labeled. Aliquots of cell lysate were immunoprecipitated with anti-paxillin antibody, an

    irrelevant mouse IgG, or anti-?4 antibody (3). Precipitates from anti-paxillin (1) or

    irrelevant IgG (2) were then extracted and re-precipitated with anti-?4 antibody. These re-precipitates (1 and 2) as well as an anti-?4 precipitate of the starting lysate (3) were

    then separated on SDS-PAGE under non-reducing conditions, and surface biotin-labeled

    polypeptides were detected as described above.

    Fig.3. The 4 tail increases the association of integrins with paxillin. a. Upper panel. Cell lysates from CHO cells stably expressing ?IIb?4?3?1A?IIb?4?3?7, or , ?IIb?6A?3?1A chimeras were precipitated with ?IIb?3-specific antibody, D-57 (anti-?IIb?3) or an irrelevant mouse IgG (IgG). Immunoprecipitated proteins were separated

    on 4 - 20% SDS-PAGE under reducing conditions, transferred to Immobilon P

    membranes, probed with biotin-labeled anti-paxillin antibody, and reacted with

    streptavidin-peroxidase followed by ECL. Two paxillin bands detected by the antibody

    probably represent the ? and ? isoforms of paxillin

    30. Lower panel. In parallel, these cell lines were surface-labeled with biotin and subjected to immunoprecipitation with D-57 or

    an irrelevant IgG. Precipitated surface proteins were separated on 4 -20% SDS-PAGE

    under non-reducing conditions, and detected with streptavidin peroxidase and ECL. b.

    Upper panel. Cell lysates from CHO cells stably expressing ?IIb?4?3?1A or ?IIb?4(Y991A)?3?1A were precipitated with D-57. Paxillin co-precipitation was

detected as described above. Lower panel. These cells were also surface-labeled with

    biotin and subjected to immunoprecipitation with D-57. Surface biotin-labeled

    polypeptides were detected as described above. The quantity of co-precipitated paxillin

    was estimated by scanning densitometry using the NIH “Image” program.

    Fig. 4. Association of the 4 tail with paxillin inhibits cell spreading. a. Equal numbers of ?IIb?4?3?1A-, ?IIb?4(Y991A)?3?1A- or ?IIb?6A?3?1A- (data not shown)-bearing CHO cells were added to Fg-coated coverslips in 24-well plates and oC incubator for 1 hr. The three cell lines expressed similar levels of

    incubated in a 37integrin ?IIb?3 as judged by flow cytometry (data not shown). The plates were washed

    with PBS twice, cells were fixed and examined by phase microscopy. Magnification =

    200 x. Photo images were taken with a Nikon Diaphot microscope equipped with a

    Sensys cooled CCD video camera. Three independent clones of ?IIb?4?3?1A and of ?IIb?4(Y991A)?3?1A-CHO cells were examined and similar results were obtained (data

    not shown). The data represent the mean ? S.D. of triplicate determinations. b. Jurkat T cell spreading on VCAM-1. ?4-null Jurkat T-cells (DMR and MHG, manuscript in

    preparation) were stably transfected with wild-type ?4 or ?4(Y991A) integrin subunit and cell lines expressing similar levels of ?4 were isolated and analyzed by flow

    cytometry (Mean fluorescence intensity ?4=58, ?4(Y991A)=49 arbitrary units). ?4- or ?4(Y991A)-expressing Jurkat T cells adherent to VCAM-1 coated coverslips were fixed

    at 1 hr and stained with rhodamine-phalloidin. Cytoplasmic protrusions in ?4(Y991A)-expressing cells were broader and more extended (arrowhead, lower panel) as compared

    with the wild-type ?4-expressing Jurkat T cells (upper panel). Cell spreading of these

Jurkat T cells was quantified using NIH “Image” software to estimate the cell area. The

    mean pixel area of ?4(Y991A)-expressing cells (897.1 pixels/cell, SEM = 25.4, n = 205)

    is significantly (p<0.001) greater than that of the wild-type ?4-expressing cells (715.8 pixels/cell, SEM = 20.3, n = 225). Cell size of the wild-type ?4- and ?4(Y991A)-expressing Jurkat T cells in suspension was not significantly different as determined by

     = 483 for wild-type ?4 cells and f = 473 for scscforward scatter flow cytometric analysis (f

    ?4(Y991A) cells). Bar equals 10 ?m. c. Mouse embryonic fibroblasts derived from

    paxillin-null (paxillin-null) or wild-type (wild-type) mice, expressing equal amounts of

    recombinant ?4 integrin, were plated on coverslips coated with VCAM-1 and incubated

    at 37oC for 1 hr. In addition, the ?4-expressing paxillin-null cells were transfected with

    vector encoding paxillin (paxillin) or empty vector (mock) together with vector encording

    GFP and plated on VCAM-1 coated coverslips. After washing, attached cells were fixed

    and GFP (+) cells were examined for spreading by phase and fluorescence microscopy.

    The data represent the mean ? S.D. Inset: 50 µg of cell lysate from mouse embryonic

    fibroblasts derived from paxillin-null (1) or wild-type (2) mice was separated on 4 20%

    SDS-PAGE and the expression of paxillin assessed by Western blot analysis using anti-

    paxillin antibody (Clone 349).

Fig.5. The 4 tail changes the kinetics of FAK phosphorylation. a. ?IIb?4?3?1A- or

    ?IIb?4(Y991A)?3?1A-expressing CHO cells were plated on petri dishes coated with 10

    ?g/ml of Fg for the indicated times. Immunoprecipitates formed with anti-FAK antibody

    were separated on 4-20% SDS-PAGE, transferred onto a nitrocellulose membrane, and

    FAK phosphorylation was detected using a mixture of PY20 and 4G10 antibodies. FAK

    phosphorylation was quantified by scanning densitometry using NIH “Image” program. Equal loading of FAK protein was verified by immunoblotting of the stripped blots with

    anti-FAK antibody (data not shown). The data represent the mean + SEM. of three independent experiments. b. ?IIb?4?3?1A- or ?IIb?4(Y991A)?3?1A-expressing CHO cells were plated on petri dishes coated with 10 ?g/ml of FN. Immunoprecipitation and

    FAK phosphorylation were performed as described in a. The data are the means of duplicate experiments.

    Fig.6. Inhibition of FA formation and enhancement of cell migration by 4 tail depends on its paxillin-binding site. a, b. CHO cells stably expressing chimeric

    integrin ?IIb?4?3?1A (?4) or chimeric integrin ?IIb?4(Y991A)?3?1A (?4(991A)) were adhered to FN or Fg coated coverslips for 45 min. After fixation and

    permeabilization, talin, F-actin and nuclei were visualized by staining with anti-talin

    mAb 8d4 followed by FITC-conjugated goat-anti-mouse (BioSource), rhodamine-

    phalloidin (Molecular Probes) and Toto-3 (Molecular Probes), respectively. Images were

    acquired using a Bio-Rad 1024 MRC laser scanning confocal imaging system. CHO cells

    expressing ?IIb?4?3?1A displayed diminished spreading, stress fibers and FAs when

    plated on Fg (a, b). Approximately 20% of the cells demonstrated a polarized

    morphology (a, arrowheads). These cells manifested lamellipodia with focal

    accumulation of talin and few stress fibers (b, arrow heads). In contrast, cells expressing

    ?IIb?4(Y991A)?3?1A spread and formed stress fibers and FAs (a, b, arrows) on Fg. CHO cells expressing ?IIb?4?3?1A or ?IIb?4(Y991A)?3?1A spread and formed stress fibers equally well when plated on FN (a). b. Higher magnification image of cells plated

on Fg-coated substrate. Talin was visualized using the mAb 8d4 with FITC-conjugated

    goat anti-mouse and F-actin was visualized using rhodamine-phalloidin. Nuclei were

    visualized with DNA dye Toto-3. Note the polarized morphology of ?IIb?4?3?1A-expressing CHO cells (top panels, arrowheads) along with diminished stress fiber and FA

    formation. This is in contrast with ?IIb?4(Y991A)?3?1A-expressing CHO cells which

    displayed well-developed FAs and stress fibers (bottom panels, arrows). Z-sections are

    5 0.3 ?m, taken at the cell-substrate interface. Bars, 15 ?m. c. Random cell migration: 10

    cells in 100 ?l DMEM plus 1% BSA were added to the upper compartment of Transwell

    chambers (8 ?m pore size) which were coated both sides with 10 ?g/ml Fg. After 3 hrs.,

    the cells that migrated to the lower side of the filter were stained and enumerated under

    200 x magnification. The presence of 20 ?g/ml of monoclonal antibody 2G12, an

    ???b?3-inhibitory antibody, inhibited more than 80% of cell migration on Fg, whereas

    the same amount of anti-VnR1, an ?v?3-inhibitory antibody, or PB1, an anti-hamster

    ?5?1-inhibitory antibody did not produce significant inhibition (data not shown). ?The

    data represent the mean ? S.D. of triplicate determinations.

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