Anti c jun antibody

Chromatin immunoprecipitation (Chip) assay was performed with EpiQuick^TM Chromatin immunoprecipitation kit (Epigentek, Farmingdale, NY)) according to the manufacturer’s manual as we previously reported^{6 (link)}. Briefly, the alginate disks containing CAF cells were exposed to 0.773 kPa for 24 h and then depolymerized with 102 mM EDTA. After being washed twice with PBS using centrifugation, the CAF cells were resuspended with the fresh culture medium containing 1% formaldehyde (final concentration) and incubated at room temperature (RT) for 10 min on a rocking platform (50–100 rpm) for fixation. The fixed CAF cells were washed three times with ice-cold PBS, lyzed with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology; Seongnam-si, South Korea), and then centrifuged to collect supernatant. The supernatant was sonicated for DNA shearing, transferred to the well coated with anti-c-Jun antibody (Santa Cruz Biotechnology, Dallas, TX), incubated at RT for 1 h. After washing PBS, proteinas K was treated and incubated at 65 °C for 90 min for DNA elution. DNA was collected using spin column, and analyzed by real-time PCR. The relative binding of c-Jun to each gene promoter was calculated using the ΔΔCt method. The results were normalized to those of input DNA^{51 (link)}.

To increase ChIP efficiency we removed soluble CSB before cross-linking DNA and proteins [8] (link), [35] (link), [58] (link). Cells were collected in Buffer B (150 mM NaCl, 0.5 mM MgCl₂, 20 mM HEPES pH 7.8, 10% Glycerol, 0.5% Triton X-100) and soluble CSB was separated from chromatin by centrifugation at 15,000 RPM for 5 min at 4°C. The resulting pellets were resuspended in Buffer B and fixed with 1% formaldehyde for 10 min at room temperature. Cross-linked cells were sonicated at 40% amplitude (30 sec on, 90 sec off, for 24 min total) using the Branson 101-135-126 Sonifier. Chromatin IP (ChIP) was performed using a monoclonal anti-CSB antibody (1B1) that recognizes the N-terminal 507 amino acids of CSB [35] (link), [39] (link) or an anti-c-Jun antibody (Santa Cruz, sc-1694). ChIP samples were reverse cross-linked in SDS sample buffer for subsequent western blot analyses [20] (link). Antibodies used for western blot analysis are rabbit anti-CSB antibodies (kindly provided by Dr. Alan Weiner, U. Washington) [35] (link), c-Jun (Santa Cruz, sc-1694) and GAPDH (Millipore, MAB374).

Cells (5 × 10⁴ each well) were seeded on four-well culture slides (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After 24 h, slides were washed in PBS, fixed with 4% paraformaldehyde for 30 min and non-specific binding was blocked using 1% BSA/PBS. Slides were then incubated with an anti-c-Jun antibody (1:40; Santa Cruz Biotechnology, Heidelberg, Germany) or anti-TUB1 antibody (1:500; Sigma-Aldrich, Steinheim, Germany), washed three times with PBS, and incubated with the AlexaFlour-anti-rabbit or AlexaFlour-anti-mouse antibody (1:150; Invitrogen, Groningen, The Netherlands). Afterwards, they were washed again and finally sealed with VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA) including 1 mg/mL DAPI (Sigma-Aldrich, St. Louis, MO, USA). Images were collected by fluorescence microscopy or confocal microscopy.