The following monoclonal antibodies were used for immunofluorescence: anti-CHC17 (2 μg ml−1; X22 (ref. 54 (link))), cortactin (1:500; Millipore, number 05–180), paxillin (1:500; Millipore, number 05–417), anti-HA (1:100; Covance, number MMS-101P). Rat anti-CD29 mAb13 against inactive β1-integrin (5 μg ml−1; BD biosciences, number BDB552828), rabbit polyclonal antisera against the conserved region of CLCs55 (link), and Hip1R (1:100; Millipore, number AB9882) were also used. Secondary labelling was done with Alexafluor-488 (1:500; Life Technologies, number A-11001) or 555-conjugated secondary antibodies (1:500; Life Technologies, number A-21422). Alexafluor 647-phalloidin (1:100; Life Technologies, number A-22287) was used to stain F-actin. The following antibodies were used for immunoblotting: anti-CHC17 (1:1,000; TD.1 (ref. 56 (link))), Hip1R (1:500; Millipore, number AB9882), β1-integrin (1:1,000; BD Biosciences, number 610467), TfR (1:1,000; BD Biosciences, number 612124), HA (1:1,000; Covance, number MMS-101P), Hip1 (1:500; Sigma, number HPA013606), anti-β-actin (1:2,000; Sigma, number A5441) and α-tubulin (1:5,000; Sigma, number T6199).
Paxillin
Manufactured by Merck Group
Sourced in United States, Italy
Paxillin is a laboratory equipment product manufactured by Merck Group. It is a multi-functional adapter protein that plays a critical role in cell adhesion and signaling processes. Paxillin serves as a scaffold for the assembly of various protein complexes, facilitating the regulation of cellular activities such as cell migration, proliferation, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
H3k27me3, by Diagenode (4 mentions)
H3k9me3, by Abcam (4 mentions)
Vinculin, by Merck Group (3 mentions)
E cadherin, by BD (3 mentions)
Phalloidin tritc, by Merck Group (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Fast protease inhibitors cocktail, by Merck Group (2 mentions)
Irdye secondary antibody, by LI COR (2 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (2 mentions)
Lamin a c, by Proteintech (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Lamin b1, by Abcam (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Lamin a c, by Merck Group (2 mentions)
Leica dmi6000, by Leica camera (2 mentions)
Donkey f ab 2, by Jackson ImmunoResearch (2 mentions)
Vectashield mounting media, by Clinisciences (2 mentions)
Setdb1, by Thermo Fisher Scientific (2 mentions)
16 protocols using paxillin
1
Immunofluorescence and immunoblotting protocols for cellular protein detection
2
Western Blot Analysis of Cellular Markers
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Cell lysis was carried out with RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with FPIC (Fast Protease Inhibitors cocktail; Sigma-Aldrich; Cat#: S8830-20TAB). The lysates were reduced in NuPAGE LDS Sample Buffer (Thermo Fisher; Cat#: NP0007) supplemented with NuPAGE Sample Reducing Agent (Thermo Fisher; Cat#: NP0009), separated by polyacrylamide gel in NuPAGE MOPS SDS Running Buffer (Thermo Fisher; Cat#: NP0001) and transferred into nitrocellulose membrane (Amersham; Cat#: 10600007). Membranes were blocked in 5% milk powder in PBST Buffer (1× PBS, 0.2% Tween 20) and incubated overnight at 4°C with the primary antibodies against SETDB1 (Abcam; Cat#: ab107225), H3 (Santa Cruz Biotechnology; Cat#: sc-8654), H3K9me3 (Abcam; Cat#: ab8898), H3K27me3 (Diagenode; Cat#: C15410069), Lamin A/C (Proteintech; Cat#: 10298-1-AP), Lamin B1 (Abcam; Cat#: ab16048), E-cadherin (BD Transduction Laboratory; Cat#: 610181), Paxillin (Millipore; Cat#: 05-417), β-actin (Sigma-Aldrich; Cat#: A5441). Membranes were incubated with the appropriate LI-COR IRDye secondary antibody (LI-COR Biosciences GmbH) and revealed by Odyssey Fc imaging system (LI-COR Biosciences GmbH). The images obtained from the slot blot assay were analyzed with the Image Studio Lite 5.2.5 software.
3
Semi-Quantitative Analysis of Podocyte β3 Integrin Activity in FSGS
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To semi-quantitatively examine the effect of FSGS patient sera on podocyte β3 integrin activity, a human podocyte cell line was cultured at 37 °C for 14 days for complete differentiation [17 (link)]. The cells were then incubated in 5% of FSGS patient serum for 24 h with lipopolysaccharide (LPS) as a positive control. Next, the cells were fixed with 4% paraformaldehyde (PFA) and processed for immunofluorescence staining for AP5 (Blood Center of Wisconsin) and paxillin (Millipore). AP5 is an antibody detecting the active state of β3 integrin by recognizing the unfolding N-terminal epitope GPNICT upon the activation of the integrin [18 (link)]. After immunostaining, confocal (Leica) images were taken to quantify the AP5 and paxillin intensity for each sample treatment. paxillin signal was used to correct AP5 signal for each treatment. The relative AP5 signal (AP5/paxillin ratio) from each patient serum was then normalized against that of normal blood donor included in each assay for final report [15 (link)]. To control for suPAR specificity, the cells were co-incubated with both FSGS sera and suPAR blocking antibody. The normalized AP5 value from normal serum treated podocytes was 1. The relative AP5 value of 1.05 or more obtained from patient serum treated podocytes was considered abnormal.
4
Multiprotein Localization Immunofluorescence
5
Immunofluorescence Staining Protocol
6
Immunofluorescence Analysis of Focal Adhesions
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Transfected HT1080 cells were plated on coverslips or on collagen (10 μg/mL) coated coverslips for 24 h. For nocodazole treatment, 10 μM nocodazole was applied for additional 1 h. Cells were fixed with 3.7% formaldehyde for 20 min, then washed three times with phosphate-buffered saline (PBS) and 0.1% Triton-X 100 in PBS for 1 min. Fixed HT1080 cells were incubated with primary antibodies Rab11 (1:100, Cell Signaling Technology), p-FAK (1:150, BD Biosciences), vinculin (1:100, Sigma, MO, USA), paxillin (1:100, Millipore, MA, USA) or Rac1 antibody (1:100, BD Biosciences) for 1 h at room temperature, and after washing three times with PBS, the HT1080 cells were incubated with an appropriate fluorescence-conjugated secondary antibody, Cy2-conjugated anti-rabbit antibody or Cy3-conjugated anti-mouse antibody, at room temperature for 1 h. The coverslips were then mounted on slides and observed using a Zeiss LSM 510 META confocal microscope (Zeiss, Germany). For vinculin and Rac1 expression level, the distribution area was the region of interest with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the expression intensities were counted and quantified.
7
Immunofluorescence Staining of Histone Modifications
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Cells were seeded on glass coverslips at 60–70% confluence, fixed in 4% paraformaldehyde, incubated with 50 mM NH4Cl to quench formaldehyde, and permeabilized with 0.5% Triton X-100. Samples were subsequently incubated overnight with primary antibody against SETDB1 (Thermo Fisher; Cat#: MA5-15722), H3K9me3 (Abcam; Cat#: ab8898), H3K27me3 (Diagenode; Cat#: C15410069), Lamin A/C (Sigma-Aldrich; Cat#: SAB4200236), E-cadherin (Abcam; Cat#: ab40772), phalloidin-TRITC (Sigma-Aldrich; Cat#: P1951), paxillin (Millipore; Cat#: 05-417) overnight at 4°C, followed by incubation with appropriate fluorophore-conjugated secondary antibodies (Donkey F(ab’)2; Jackson ImmunoResearch Laboratories) for an additional 1 h. Cell nuclei were stained with DAPI (Life Technologies; Cat#: 62248). Coverslips were mounted with Vectashield mounting media (Clinisciences). Microscopy was performed using an inverted microscope Leica DMI-6000, using 40×, 63× or 100× immersion objectives. Images were taken with the HQ2 Coolsnap motorized by MetaMorph 7.10.2.240 software. All images were processed with ImageJ (Fiji) software.
8
Immunofluorescence Staining of Focal Adhesions
9
Comprehensive Immunofluorescence Staining Protocol
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Following primary antibodies were used in this study: lamin B (Santa Cruz cat. no. sc-6217); filamin (Santa Cruz cat. no. sc-28284); alpha-actinin-4 (Abcam cat. no. ab96866); spectrin (Sigma Aldrich cat. no. S1390); paxillin (Millipore cat. no. 05-471); vinculin (Sigma Aldrich cat. no. V4505); mDia1 (BD Biosciences cat. no. P66520-050); SUN2 (Abcam cat. no. ab124916); emerin (Abcam cat. no. ab40688); Arp3 (Welch et al. 1997 (link)); cofilin (Abcam cat. no. ab11062); P-cofilin (Cell Signaling cat. no. 3313); Arp6 (Sigma Aldrich cat. no. R35554); Arp5 (Kitayama et al. 2009 (link)); Arp8 (Aoyama et al. 2008 (link)); Brg1 (Abcam cat. no. ab70558); hnRNP U (Santa Cruz, clone 3G6); H3K9Me2 (Millipore cat. no. 17-648); H3K4Me2 (Millipore cat. no. 07-030); CTD-phosphoS2 (Abcam cat. no. ab24758); RPA194 (Santa Cruz, cat. no. sc-28714); and BrdU (Sigma Aldrich, clone BU-33).
Secondary antibodies used in this study are donkey anti-rabbit IgG conjugated with Alexa Fluor 568 (A10042), goat anti-mouse IgG conjugated with Alexa Fluor 647 (A21236) and donkey anti-goat IgG cojugated with Alexa Fluor 647 (A21447) all purchased from Life Sciences.
Secondary antibodies used in this study are donkey anti-rabbit IgG conjugated with Alexa Fluor 568 (A10042), goat anti-mouse IgG conjugated with Alexa Fluor 647 (A21236) and donkey anti-goat IgG cojugated with Alexa Fluor 647 (A21447) all purchased from Life Sciences.
10
Immunofluorescence Staining of Cell Lines
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Cells were seeded on glass coverslips at 60 -70 % confluence, fixed in 4 % paraformaldehyde, incubated with 50 mM NH4Cl to quench formaldehyde, and permeabilized with 0.5 % Triton X-100. Samples were subsequently incubated overnight with primary antibody against SETDB1 (Thermo Fisher; Cat#: MA5-15722), H3K9me3 (Abcam; Cat#: ab8898), H3K27me3 (Diagenode; Cat#: C15410069), Lamin A/C (Sigma-Aldrich; Cat#: SAB4200236), E-cadherin (Abcam; Cat#: ab40772), phalloidin-TRITC (Sigma-Aldrich; Cat#: P1951), paxillin (Millipore; Cat#: 05-417) overnight at 4 °C, followed by incubation with appropriate fluorophore-conjugated secondary antibodies (Donkey F(ab')2; Jackson ImmunoResearch Laboratories) for an additional 1 hour. Cell nuclei were stained with DAPI (Life Technologies; Cat#: 62248). Coverslips were mounted with Vectashield mounting media (Clinisciences). Microscopy was performed using an inverted microscope Leica DMI-6000, using 40x, 63x or 100x immersion objectives. Images were taken with the HQ2 Coolsnap motorized by MetaMorph 7.10.2.240 software. All images were processed with ImageJ (Fiji) software.
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