Rabbit anti igg

ChIP assays were performed with the ChIP assay kit (Millipore). In brief, cells were cross-linked with 1% formaldehyde for 10 min at room temperature, lysed with SDS buffer, and DNA was sheared by sonication. Samples were diluted and pre-cleared with protein A-agarose/salmon sperm for 30 min at 4 °C. DNA-protein complexes were incubated with 1:5,000 rabbit anti-KLF5 antibody [21 (link)] at 4 °C overnight and precipitated with protein A-agarose for 1 h. DNA was purified with the Qiaquick PCR purification kit (Qiagen), and quantitative PCR on the TP53 promoter using specific primers (Fig. S1) was performed with Power SYBR Green Master Mix (Life Technologies). For ReChIP, the first round of ChIP was performed as for ChIP using an anti-KLF5 antibody (#21017-1-AP, Proteintech) and rabbit anti-IgG (#8726 S, Cell Signaling Technology). After washing, the complex was incubated for 30 min at 37 °C in 75 μL TE/10 mM DTT, eluted, and then diluted 20x with ChIP dilution buffer. A second round of ChIP was performed overnight at 4 °C with rotation using anti-SIN3A or anti-HDAC2 antibody on KLF5 IP samples or anti-IgG antibody on IgG samples, as per the manufacturer protocol.

For Co-IP assays, HEK-293T cells were transfected as indicated: 36 h later, cells were lysed in IP buffer (1% Triton X-100, 150 mM NaCl, and 50 mM Tris/HCl (pH 7.5), 1 mM PMSF, and protease inhibitor cocktail) and incubated with anti-Flag M2 affinity gels for 4 h at 4 °C. For reverse IP, HEK-293T cells were lysed and incubated with anti-HA antibody and protein A/G agarose for 12 h at 4 °C. Agarose was washed five times with IP assay buffer, boiled in SDS sample buffer, and subjected to Western blot analysis. Western blotting and immunofluorescence (IF) were performed as previously [40 (link)].
The following antibodies were used: mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-Flag (F7425, Sigma-Aldrich), rabbit anti-GST (2622, Cell Signaling Technology), mouse anti-β-actin (66009-1-Ig, Proteintech), rabbit anti-Snail (3879, Cell Signaling Technology), mouse anti-Snail (sc-271977, Santa Cruz), rabbit anti-p66β (ab76925, Abcam), anti-biotin, HRP-linked (7075, Cell Signaling Technology), and rabbit anti-IgG (2729, Cell Signaling Technology), mouse anti-α-tubulin (66031-1-Ig, Proteintech), rabbit anti-Fibronectin1 (15613-1-AP, Proteintech), rabbit anti-E-cadherin (20874-1-AP, Proteintech), rabbit anti-Vimentin (10366-1-AP, Proteintech).

HEK293 cells were transfected using Lipofectamine 3000 according to the manufacturer’s instructions and were incubated for at least 48 hr. Cells were harvested and proteins were extracted in lysis buffer containing 25 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 1% Igepal, 5% Glycerol, 1 x EDTA-free proteinase inhibitor, 0.5 mM DTT and 2.5 U/mL Benzonase. Protein A magnetic beads were washed 2 x with PBS including 0.02% Tween-20 and were incubated on a rotating wheel in RT for 2 hr with 2 μg of the following antibodies suspended in 200 μL 2 x with PBS/0.02% Tween-100: rabbit anti-IgG (Cell Signal), rabbit anti-flag (Sigma-Aldrich) or rabbit anti-C1ql2 (Sigma-Aldrich). Beads were washed 2 x with PBS/0.02% Tween-20, resuspended in 50 μL 2 x with PBS/0.02% Tween-20 and 40 μg protein extract was added. Beads were incubated o/n on a rotating wheel at 4 °C. Beads were thoroughly washed with 2 x with PBS/0.02% Tween-20, resuspended in 2 x SDS loading dye, and boiled for 10 min at 95 °C. Western blot (see above) was performed. Briefly, membranes were blocked with 5% non-fat milk (Sigma-Aldrich) and incubated with mouse anti-flag M2 (1:2000; Sigma-Aldrich). Three independent experiments were carried out.

Rabbit anti-phosphorylated and anti-total p38MAPK mAb, rabbit anti-phosphorylated and anti-total IκB mAb, rabbit anti-phosphorylated and anti-total AKT mAb, rabbit anti-Mcl-1 mAb, rabbit anti-procaspase 3 mAb, mouse anti-active caspase-3 mAb, mouse anti-β-actin mAb, rabbit anti-IgG, and mouse anti-IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-His mAb was purchased from Affinity Biosciences, Inc (OH, USA). The rabbit anti-C. psittaci mAb was a gift from Professor G. Zhong (University of Texas Health Science Center at San Antonio, TX). The rabbit anti-SOD2 antibody, mouse anti-Bax antibody was purchased from ImmunoWay Biotechnology Company (Plano, TX, USA). The inhibitors SB203580, BAY117082, LY294002, staurosporine (STS), and Cy2-conjugated goat anti-rabbit IgG, DAPI, Cy3-conjugated goat anti-mouse IgG, and Cy3-conjugated goat anti-rabbit IgG and mouse anti-NF-κB p65 antibody were obtained from Abcam (Cambridge, MA, USA), recombinant human IL-8 from (Biolegend, CA, USA), anti-IL-8 mAb was obtained from GeneTex, USA.