AHO0492

antibody from Invitrogen Antibodies

Targeting: PXN

Provider product page for AHO0492

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [20]
Comments [0]
Validations
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [14]

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Product number: AHO0492 - Provider product page
Provider: Invitrogen Antibodies
Product name: Paxillin Monoclonal Antibody (5H11)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: Staining of formalin-fixed, paraffin-embedded tissues requires boiling tissues in 1mM EDTA, pH 8.0, for 10-20 min followed by cooling at room temperature for 20 min. Recommended positive controls include HeLa cells and colon carcinoma.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 5H11
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

PXN protein structure - AHO0492 shown in red.

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: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. ( A ) Survival outcome analysis in NSCLC patients with wild-type versus mutated PXN. Left and right graphs show overall and individual mutant survival curves, respectively. The relative expression of PXN, p-PXN, BCL-2, FAK, phospho-FAK, Cox IV, DRP-1, and MFN-2 was evaluated by IHC on two sets of samples; seven NSCLC patient samples bearing wild-type PXN and six NSCLC patient samples that were identified to have various PXN mutations as shown in ( B ). ( C and D ) represent the averaged staining intensity scores among all WT and all mutants. ( E ) Shows graph of staining intensity scores of the proteins assayed as expressed in the cytoplasm of the cells. ( F )The panel shows photographs of IHC stains represented in one wild-type and one mutant sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Effect of HGF stimulation on HEK-293 wild-type PXN and mutants shown in ( A ) confocal images after treatment with HGF and stained with MitoTracker Red and Hoechst and ( B ) measurements of mitochondrial functions by Imaris.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Recruitment of multiple FA components is defective in ItgalphaV- and Itgbeta1-KD ItgV-KD cells. A ) Control, Itgbeta1- and ItgalphaV-KD MDCK cells were trypsinized, seeded onto Col I-coated coverslips in serum-free culture medium, allowed to settle for 75 minutes, fixed and stained for actin (TRITC-phalloidin, red), nuclei (DAPI, blue) and beta1-integrin (green), B ) talin, C ) paxillin, D ) vinculin or E ) zyxin. F ) Control, Itgbeta1- and ItgalphaV-KD MDCK cells expressing GFP-ILK (green) were treated as above and stained for actin (TRITC-phalloidin, red) and nuclei (DAPI, blue). Scale bars are 20 um in all figure panels.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Paxillin in native human A431 cells using a Paxillin monoclonal antibody (Product # AHO0492).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Nicotinic acid (NA) post-transcriptionally downregulates paxillin. U251 cells were treated with the indicated concentration of NA for 4 h. (A-F) DAPI labeling (blue) for nuclei, rhodamine phalloidin labeling for F-actin (red) and immunocytochemistry for p-paxillin (green) were carried out as described in Materials and methods. Regions denoted with rectangles are amplified and shown in insets with red and green channels merged. (G) Western blot analyses for whole-cell lysates were performed with an anti-paxillin antibody. Membranes were stripped and reblotted for p-paxillin and beta-actin. (H) Total RNA was extracted, and semi-quantitative RT-PCR was carried out for paxillin mRNA. GAPDH was included as a loading control; -RT, control with no reverse transcriptase (same below). (I) Ectopic expression of 2.5 ug of paxillin in U251 cells, significantly increased paxillin mRNA.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 FAK-paxillin interaction conditions FAK localization to FA. ( A ) Cell extracts from A375 melanoma cells transiently co-transfected with siFAK and wild-type FAK-GFP or FAK-I936/I998-GFP were analyzed by immunoprecipitation (IP) using anti-FAK Ab and blotted for paxillin and FAK. The expression level of proteins in the corresponding cell lysate is shown. ( B ) Representative Western blot showing A375 melanoma cells transfected as described above and blotted for P-Y397 FAK, FAK total, eGFP and actin. ( C ) Cells transiently co-transfected with siFAK and wild-type FAK-GFP or FAK-I936/I998-GFP were fixed and labelled for paxillin (purple), actin (red) and P-Y397 FAK (cyan). Scale bar: 10 um. ( D ) Histograms represent the mean normalized to control +- SD of FAK localization in melanoma cells. Signals were measured by reporting the P-FAK signal in FA over that in the cytoplasm from 3 independent experiments ***, p < 0.001; unpaired t -test compared to control condition.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure S4. Growth factor activation at FAs. (A) HeLa cells FCS starved for 18 h (-FCS) or starved and stimulated with EGF or IGF for 10 min were analyzed by immunofluorescence (left) and immunoblotting (right). (B) Cells were starved and stimulated with IGF, EGF, or amino acids. FAs remaining on coverslips after removal of cell bodies were immunostained using paxillin and p-IGFR1, p-EGFR, or SLC3A2 antibodies. (C) Cells were starved and stimulated as indicated and immunostained for p-EGFR, p-IGFR1, SLC3A2, and LAMP1, and colocalization at the cell periphery (Manders coefficient) was quantified. (D and E) Interaction network analysis of growth factor receptors and mTOR-associated proteins (D) and lysosome-associated proteins (E) detected in adhesion complex proteomes (meta-adhesome datasets). Proteins (nodes) are annotated with gene names for clarity. Error bars represent SEM; n = 3 independent experiments. Scale bars, 20 um (insets, 10 um). Nuclei were visualized with DAPI; original localization of nuclei in FA preparations (B) is indicated by dashed circles. For C, ***, P < 0.001; two-sided Student's t test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Plexin-B2 influences cell-matrix adhesion and controls contractility in hESC colonies. a , b TIRF microscopy images and quantification show altered number, size, and shape of focal adhesions (FAs) in PLXNB2 mutant hESC colonies ( a ) and hNPCs ( b ) compared to wild-type (WT) cells. Cells were stained for FA marker paxillin and F-actin (phalloidin). Enlarged images of boxed areas are shown below in inverted black and white, with dashed lines outlining individual cells. Box plots depict 25-75% quantiles, minimal and maximal values (whiskers), median (midline), and mean (cross signs). For hESCs: FA number, n = 14-15 cells per group. FA area, n = 2271 FAs for WT, n = 1042 for PB2 KO, and n = 978 for PB2 OE. FA circularity, n = 1042 FAs for WT, n = 1042 for PB2 KO, and n = 978 for PB2 OE. * P = 0.0397, *** P < 0.0001. For hNPCs: FA number, n = 20 cells per group. FA area and circularity, n = 1829 FAs for WT, n = 2385 for PB2 KO, and n = 4528 for PB2 OE. Kruskal-Wallis test followed by Dunn's multiple comparisons test. ** P = 0.0084, *** P < 0.0001. c Molecular dynamics mathematical simulation. Left, components of simulation based on a bead-spring model. The tip of the simulated AFM probe is depicted as a green sphere, with elastic constants K x and K y in the x and y directions, respectively. Right, graphs of force as a function of distance traveled by the simulated AFM probe in two different scenarios. Left, when varying the actin-membrane attraction energy that simulates

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Cell-Matrix Adhesion Area of PCs (dif31) Placed on PDMS Micropillar Arrays of High (137 kPa) and Intermediate (47.2kPa) Stiffness (A-F) Confocal immunofluorescence images of PCs (dif31) seeded on PDMS micropillar arrays of two different stiffness (137 and 47.2 kPa), functionalized with FN (blue). Cells were stained for vinculin (A and B), paxillin (C and D), and talin (E and F) (green). (G) Average cell-matrix adhesion area of PC cells on PDMS micropillar arrays with 137 kPa stiffness (right) and 47.2 kPa (left), after 4 h of incubation. All error bars are SEM derived from two independent experiments performed with a minimum of two replicates. At least 30 cells were analyzed from each sample. NS, p > 0.05, * p < 0.05, ** p < 0.005, *** p < 0.0005 according to the Mann-Whitney test. See also Figure S3 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 3 Stress fibers and focal adhesions increase in Bis-T-23-treated HPCs. (A) Double-immunofluorescence of V5-tagged dynamin 2 (Dyn2)-WT or K562E (bottom panels) and actin filaments (top panels) in Bis-T-23-treated (50 muM) HPCs and corresponding solvent controls (DMSO). Scale bar: 20 mum. (B) Stress fiber density in Bis-T-23-treated dynamin 2-WT- or K562E-expressing HPCs. Data are means +- S.E.M. of 35 cells (dynamin 2-WT/DMSO), 33 cells (dynamin 2-WT/Bis-T-23), 38 cells (dynamin 2-K562E/DMSO) or 35 cells (dynamin 2-K562E/Bis-T-23) from three independent experiments. (** p < 0.01). (C) Double-immunofluorescence of V5-tagged dynamin 2-WT or K562E (bottom panels) and paxillin (top panels) in Bis-T-23-treated (50 muM) HPCs and corresponding solvent controls (DMSO). Scale bar; 20 mum. (D) Number of focal adhesions in Bis-T-23-treated dynamin 2-WT- or K562E-expressing HPCs. Data are means +- S.E.M. of 35 cells (dynamin 2-WT/DMSO), 32 cells (dynamin 2-WT/Bis-T-23), 35 cells (dynamin 2-K562E/DMSO) or 34 cells (dynamin 2-K562E/Bis-T-23) from three independent experiments. (** p < 0.01). more than 32 cells from three independent experiments. (** p < 0.01).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Pericytes Exhibit a Relaxed Phenotype in Response to CRC-Secreted Factors CRC-derived factors promote pericyte relaxation and changes in cell-matrix interactions. (an i-iv) Representative images of pericytes stained for phosphorylated myosin light chain (green, iii. ), paxillin (orange, iv. ), and cell nuclei (blue, ii.) after three days of culture in conditioned media. (B) Fluorescence quantification of p -MLC. Phosphorylation of myosin light chain decreases significantly in pericytes exposed to HCT116 CM as compared to control conditions. (C) Fluorescence quantification of paxillin. No significant change in paxillin expression was observed for any condition, despite visual differences in staining patterns. Scale bar 20 mum. Significance: ***p < 0.001. Data are represented as mean +- SEM

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Nup93 modulates triple-negative, claudin-low BCC migration through actin cytoskeleton (AC) remodeling. (A, B, C, D, E, F) NUP93 KD induces dramatic changes to the cytoskeleton, including thinning of the cortical actin layer (A, B) ( t test with P < 0.05, N >= 8 independent regions in three biological replicates, data normalized to ctrl cells), increase in paxillin foci (C, D) ( t test with P < 0.001, N >= 4 independent regions in three biological replicates, data normalized to ctrl cells), and formation of a dense network of actin stress fibers (E, F) (details of single cells). Images and intensity profiles are representative of each specific condition. Scale bars: 10 mum for (A, C) and 5 mum for (E, F). (G, H) Partial rescue of Nup93 limits the expression of the stress fiber-related protein LIMCH1. Representative images and intensity profiles. Scale bars: 10 mum. (I, L) NUP93 KD impairs breast cancer cell 3D migration. (I, J, K) Schematic of the microfluidic assay for 3D single cell tracking (I) and quantification of cell migration (J) ( t test with P < 0.001, N >= 100 single cells tracked per condition in three biological replicates, data normalized to ctrl cells) and adhesion (K) ( t test with P = 0.07, N >= 25 independent regions in three biological replicates, data normalized to ctrl cells). (L) Rescue of Nup93 partially restored cell migration (L) (ANOVA test with P < 0.05 [*] and P < 0.01 [**], N >= 120 single cells tracked per condition in three biological r