Anti paxillin antibody

WT and paxillin-overexpressing HSCs were labeled with anti-paxillin antibody (cat. number 610051, BD Biosciences, Franklin Lakes, NJ) at a dilution of 1:200. Z-stack images of individual cells were taken using a 60× objective lens on an Olympus Fluoview Fv10i laser scanning confocal microscope, and quantitative analysis of focal adhesions was performed as described previously (Horzum et al., 2014 (link)). In brief, images were converted into 16-bit and a Z-projection with maximum intensity was performed. Subsequently, the following commands were executed using FIJI software: ‘subtract background with a rolling ball radius of 50 pixels’, enhance contrast by running CLACHE plug-in with block size=19, histogram bins=256, and maximum slope=6’, ‘minimize background by applying mathematical exponential (exp)’, and ‘adjust brightness and contrast automatically’. Then, a Mexican Hat filter was applied (http://bigwww.epfl.ch/sage/soft/LoG3D) with a radius of 5. Next, images were converted to binary using the threshold command and finally, the ‘analyze particles’ command was executed and quantitative statistics were collected (e.g. size, area, number of focal adhesions, and % area occupied by focal adhesions).

Non-transfected EC cells grown on fibronectin coated coverslips were fixed with cold 4% PFA for 15 min at RT and washed 3× 5 min with PBS. Unspecific binding was blocked with 10% FBS/PBS for 1h at RT. Anti-paxillin antibody (BD Biosciences, San Jose, CA, USA, #610619) was diluted in 1% FBS/PBS and incubated overnight at 4 °C. Cells were washed 3× 5 min with PBS and the incubated with donkey anti-mouse AlexaFluor 594 (Invitrogen, Waltham, MA, USA, www.Thermofisher.com) secondary antibody for 1 h at 4 °C, followed by 3× 5 min washes with PBS and PBS with 1 μg mL⁻¹ Hoechst 33342 for 15 min, followed by a quick wash and mounting on an objective glass. Confocal images of EC that had spread were taken and analyzed by ImageJ for fluorescence intensity in the peripheral area vs. the perinuclear area. The boundary between the perinuclear and peripheral was set at half the distance from the nucleus to the edge of the cell and the fluorescence intensity in each region determined. Ratios were calculated for each cell (10 wild type and 8 KO).

MM cells at a confluence of 70–90% were washed with ice-cold PBS and lysed with 1 ml ice-cold Triton X-100 lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0) supplemented with protease and phosphatase inhibitors. Cell lysates were incubated on ice for 30 min and then centrifuged (10,000×g, 10 min at 4 °C). To pull down CR, 2–4 μg of a rabbit polyclonal anti-CR antiserum (CR7699/4; Swant) was added to the cleared lysate and incubated for 10 min at 4 °C. μMACS protein A MicroBeads suspension (100 μl; Miltenyi Biotec, Auburn, CA, USA) was added to the lysate and incubated at 4 °C for 30 min. Samples were loaded on MACS separation columns (Miltenyi Biotec) and subjected to magnetic immunoprecipitation. The columns were washed 3 times with a wash buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0). Bound protein complexes were eluted in 50 μl of pre-warmed SDS gel loading buffer 1X (50 mM Tris-HCl, pH 6.8, 50 mM DTT, 1% SDS, 0.005% bromophenol blue, 10% glycerol) and subjected to Western Blotting for septin 7 as described above. A mouse monoclonal anti-Paxillin antibody (1:2000; BD Bioscience) was used as negative control.

The findings in this report rely heavily on the double-staining of cultured cells with Rhodamine (Rho)- or fluorescein (FITC)-phalloidin (1:40 and 1:10, respectively; Molecular Probes) and one of the following antibodies: anti-sarcomeric-α-actinin (s-α-actinin) antibody (1:2500, clone EA-53, Sigma), anti-β-actin (1:800, clone AC-15, Sigma), anti-titin Z1Z2 antibody (1:30, [38 (link)]), anti-C-protein (1:50, clone MF-1) that stains the A-bands of striated myofibrils [39 (link)], anti-nebulin M177M181 antibody (1:50, [40 (link)]), anti-desmin antibody (1:200, Sigma), anti-tyrosylated-α-tubulin antibody (undiluted, clone YL1/2), anti-vinculin antibody (1:50, clone VIN-11-5, Sigma), anti-paxillin antibody (1:50, BD Transduction Labs.), anti-β1-integrin antibody (1:10, Sigma), anti-talin antibody (undiluted, clone 8e6, Iowa Hybridoma Bank), anti-cadherin 2 (N-cadherin) antibody (1:5, BioGenex), anti-Mrf4 (Myf6) antibody (1:100, Santa Cruz Biotechnology), anti-Arp2/3 antibody (1:100, Abcam), and anti-vimentin antibody (1:100, clone Vim 3B4, Boehringer Mannheim).

Anti-MEKK2 and ERK2 antibodies were purchased from Santa Cruz Biotechnology. Anti-FLAG antibodies were purchased from Sigma. Protein G agarose beads were purchased from Roche Applied Science. Anti-paxillin antibodies were purchased from BD Biosciences. Anti-phospho-MKK4 antibodies were purchased from Cell Signaling Technology. Recombinant MEKK2 was purchased from SignalChem, and recombinant paxillin was purchased from RayBiotech, Inc.