The antibodies and inhibitors used are as follows: active caspase 3, poly ADP ribose and vimentin (R&D Systems); Akt, Akt-pS473, ErbB2, ErbB2-pY1248, ErK-pT202/T204, FAK-pY925 and Src-pY416 (Cell Signaling); FAK-pY397, FAK-pY576, FAK-pY577 and paxillin-pY31 (Invitrogen); E-cadherin and paxillin (BD Transduction Laboratories); anti-V5 (Serotec); FAK and N-cadherin (Santa Cruz); actin and tubulin (Sigma); FAK pY407, FAK pY861 and paxillin pY118 (Biosource); secondary antibodies (Jackson Laboratory); Hoechst 33528 (Sigma). The FAK inhibitor AZ675 was provided by AstraZeneca (Alderly Edge).
Paxillin
Manufactured by BD
Sourced in United States
Paxillin is a cytoskeletal protein that functions as an adapter, linking cell surface receptors to the actin cytoskeleton. It plays a role in the regulation of cell adhesion, motility, and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Vinculin, by Merck Group (6 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (6 mentions)
E cadherin, by BD (5 mentions)
α tubulin, by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Talin, by Merck Group (4 mentions)
Vimentin, by Merck Group (4 mentions)
Ps473 akt, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
N cadherin, by BD (3 mentions)
Gapdh, by Merck Group (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
β actin, by Merck Group (3 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Py397 fak, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Cdc42, by BD (2 mentions)
46 protocols using paxillin
1
FAK Inhibitor Effects on Cell Signaling
2
Quantitative Immunoblotting with LI-COR
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Immunoblotting was performed as described by Zhang et al. (2012) , with the exception that proteins were transferred to nitrocellulose membranes (Invitrogen). Signal was detected with fluorescence-linked secondary antibodies on a Li-Cor Odyssey infrared imager. Primary antibodies were NMHC 2A (1:50,000; BioLegend), NMHC 2C (1:5000; BioLegend), GFP (1:5000; Abcam), p-paxillin (1:1000; BD Transduction Laboratories), paxillin (1:20,000; BD Transduction Laboratories), and GAPDH (1:5000; Meridian).
3
Immunoblot Analysis of Signaling Proteins
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Immunoblot analyses were carried out as described previously.32 , 39 The detailed protocols are described in Appendix S1 . Antibodies against the following proteins were purchased. ErbB3 (C‐17), DOCK180 (H4), C3G (C19), Gab1, CRKL (C20), and CD63 (MX‐49.129.5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and paxillin, p130Cas, and CRK were from BD Transduction Laboratories (Lexington, KY, USA). Epidermal growth factor receptor (EGFR), phospho‐EGFR (Y1148), c‐Met, phospho‐c‐Met (Y1234/1235), ErbB2 (29D8), focal adhesion kinase (FAK), phospho‐FAK (Y397), phospho‐Gab1 (Y307), Src, phospho‐Src (Y416), ERK1/2, phospho‐ERK1/2, Akt, and phospho‐Akt (S473) were from Cell Signaling Technology (Beverly, MA, USA). Actin was from Chemicon International (Temecula, CA, USA) and α‐tubulin was from Sigma‐Aldrich.
4
Podocyte Cytoskeleton and Focal Adhesion Analysis
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PACSIN2-wt or S313E/A were transiently transfected into proliferating human podocytes. Cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100 and stained when mentioned with CellMask Blue (H32720, Thermo Fisher Scientific, Waltham, MA, USA), Hoechst (33342, Merck), anti-β-tubulin III IgG (T2200, rabbit polyclonal, AB_262133, Merck), phalloidin-488 (1:250, A12379, Thermo Fisher Scientific) to stain filamentous actin (F-actin), c-myc (M4439, mouse monoclonal, Merck,) and paxillin (610051, mouse monoclonal; BD Transduction laboratories, Franklin Lakes, NJ, USA) to stain focal adhesions. Alexa Fluor 594 anti-rabbit IgG (A-21207, donkey polyclonal, Invitrogen) and Alexa Fluor 488 anti-mouse IgG (A21202, donkey polyclonal, Thermo Fisher Scientific)) were used as secondary antibodies. Imaging was carried out using the Opera Phenix HCS system (PerkinElmer, Waltham, MA, USA) with a 20× air objective (NA 0.4), followed by processing with CellProfiler 3.1.8 (https://cellprofiler.org/ accessed on 10 January 2023) [16 (link)] to correct for non-uniform illumination, detect the cells or focal adhesions and extract numerical features.
5
Western Blot Analysis of Cell Signaling
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After cells were treated with F806 for 24 hr, total cell lysates were prepared in Laemmli sample buffer (Bio-Rad, 101-0737, Hercules, CA, USA). Western blots were performed as described previously [44 (link)]. Primary antibodies to ERK1/2, α5 integrin, β4 integrin and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while antibodies to PARP, phospho-ERK1/2 (p-ERK1/2, Tyr204), phospho-AKT (p-AKT, Ser473) and AKT were purchased from Cell Signaling Technology (Danvers, MA, USA), along with phospho-FAK (p-FAK, Tyr397) from Invitrogen (Camarillo, CA, USA), Kindlin-2 from OriGene Technologies (Rockville, MD, USA), FAK, paxillin, and β1 integrin from BD Transduction Laboratories (Franklin Lakes, New Jersey, USA). Appropriate HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA). Detailed information about the antibodies is listed in Supplementary Table 2 .
6
Quantitative Analysis of Protein Interactions
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For immunoprecipitation studies, equal amounts of protein were used from ex vivo epithelial explants extracted in TX/OG from different days in culture or between treated and untreated samples. For immunoprecipitations, samples were incubated at 4°C sequentially with primary antibody and TrueBlot Immunoprecipitation beads (eBioscience, San Diego, CA) and the immunoprecipitates subjected to SDS/PAGE as described previously and earlier (Walker et al., 2002 (link)). Antibodies used included vimentin (Developmental Studies Hybridoma Bank) and paxillin (BD Transduction Laboratories). Immunoblots were scanned and densitometric analysis was performed using Kodak 1D software. To standardize these results for the paxillin IP, quantitative analysis was performed. For each protein analyzed, the densitometry results were normalized to D1 OAZ, and then the ratio of each coprecipitated protein to the original immunoprecipitated protein was calculated for each sample. For vimentin IP, the ratio of myosin IIA versus myosin IIB associated with vimentin was calculated for each sample.
7
Immunofluorescence Staining of Cell Adhesion
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The antibodies used for immunofluorescence were against E-cadherin (13-1900, Invitrogen and 610182, BD Transduction Laboratories), N-cadherin (610921, BD Transduction Laboratories), ZO-1 (617300, Invitrogen), fibronectin (F-3648, Sigma-Aldrich), paxillin (610052, BD Transduction Laboratories), GFP (chicken, Aves Labs), SATB2 (Abcam, ab34735), beta-actin (C4, sc-47778, Santa Cruz), FBXO32 (sc-33782, Santa Cruz) (ab168372), ZEB1 (sc-25388 (H-102) Santa Cruz), ATF-3 (sc-188 (C-19) Santa Cruz), Cleaved caspase 3 (Asp175, 9661 S, cell signaling), FLAG tag M2 (F1804, Sigma), HA tag monoclonal antibody (c15200190, Diagenode), Myc tag (ab9106, Abcam), Ub mouse monoclonal (sc8017, Santa Cruz), Lamin B (sc-6216 (C-20), Santa Cruz), Histone H3 tri methyl K4, (ab12209, Abcam), Histone H3 tri methyl K9, (ab8898, Abcam), Histone H3 acetyl K27, (ab4729, Abcam), Alexa Fluor-488 goat anti-mouse IgG (H + L) (A11029, Invitrogen); Alexa Fluor-568 goat anti-rabbit IgG (H + L) (A11011, Invitrogen), Alexa Fluor-633 goat anti-rat IgG (H + L) (A21094, Invitrogen), and Alexa Fluor-633 phalloidin (A22284, Invitrogen), which was used to stain F-actin.
8
Microscopic Analysis of Cell Adhesion
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Antibodies for talin (Sigma-Aldrich), vinculin (Sigma-Aldrich), liprin-α1 (Proteintech), cortactin (clone 4F11, Millipore), phalloidin AlexaFluor-594 and AlexaFluor-488 (Life Technologies), activated β1-integrin (12G10, Abcam), total β1-integrin (BD Transduction Laboratories), paxillin (BD Transduction Laboratories), pFAK (BD Transduction Laboratories), vimentin (Sigma-Aldrich), N-cadherin (Abcam), b-actin (Santa Cruz), MT1-MMP (Millipore), Rab11 (BD Transduction Laboratories), EEA1 (BD Transduction Laboratories), PKCε (BD Transduction Laboratories) and E-cadherin (BD Transduction Laboratories) were used in immunofluorescence and western blotting. Phalloidin-TRITC and collagen type I from rat tail (Sigma-Aldrich) were used in invasion assays. Secondary antibodies goat anti-mouse AlexaFluor-488 and goat anti-rabbit Alexa Fluor-594 (Life Technologies) were used in 1:400 dilution in immunofluorescence. For immunoblotting, secondary antibodies HRP-Goat Anti-Mouse IgG (H + L) and HRP-Goat Anti-Rabbit IgG (H + L) (Life Technologies) were used in 1:10000–20000 dilution.
9
Quantifying Focal Adhesion Signaling
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Primary antibodies: pY410CAS (rabbit; Cell Signaling), pY165CAS (rabbit; Cell Signaling), pY118paxillin (rabbit; Sigma), paxillin (mouse, BD Transduction Laboratories), pY397FAK (rabbit; Invitrogen), pY861FAK (rabbit; Abgent), pY529Src (rabbit; Sigma), GFP (mouse; Invitrogen), actin (goat; Santa Cruz Biotechnology). Secondary antibodies (conjugated with horseradish peroxidase): goat anti-rabbit (Thermo Scientific), donkey anti-goat (Santa Cruz Biotechnology), rabbit anti-mouse (Abcam). AlexaFluor goat anti-mouse 594 (Molecular Probes) as a secondary antibody for immunofluorescence. For 3D STED imaging, Abberior STAR 635 (goat anti-rabbit) and Abberior STAR 580 (goat anti-mouse) were used as secondary antibodies.
10
Western Blot Antibody Library
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Antibodies for western blot detection included actin (I-19, Santa Cruz Biotechnology, cat. n. sc-1616, RRID: AB_630836 ), Akt (Cell Signaling Technology, cat. n. 9272, RRID: AB_329827 ), Akt pS473 (Cell Signaling Technology, cat. n. 9271, RRID: AB_329825 ), Cas (BD Transduction Laboratories, cat. n. 610271, RRID: AB_397666 ), Cas pY410 (Cell Signaling Technology, cat. n. 4011, RRID: AB_2274823 ), Erk1/2 (Promega), phospho-Erk1/2 (Promega, cat. n. V1141, RRID: AB_430839 ), FAK (C-20, Santa Cruz Biotechnology, cat. n. sc-558, RRID: AB_2300502 ), FAK pY397 (Invitrogen, cat. n. 44–624G, RRID: AB_2533701 ), FAK pY861 (Abgent, cat. n. AJ1285f, RRID: AB_10818227 ), paxillin (BD Transduction Laboratories, cat. n. 610052, RRID: AB_397464 ), paxillin pY118 (Cell Signaling Technology, cat. n. 2541, RRID: AB_2174466 ), Src (clone 184Q20, Invitrogen, cat. n. AHO1152, RRID: AB_2536324 ), Src pY418 (Cell Signaling Technology, cat. n. 2101, RRID: AB_331697 ), Stat3 (C-20, Santa Cruz Biotechnology, cat. n. sc-482, RRID: AB_632440 ), and Stat3 pY705 (Cell Signaling Technology, cat. n. 9131, RRID: AB_331568 ).
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