Western lightning plus ecl solution

Sample preparation for SDS-PAGE and immunoblot analysis were performed as described previously [38 (link)]. A total of 6, 10 or 14 µg cellular protein was applied per lane. Polyclonal sera against σ^S, σ^FliA and CsgD (custom-made by Pineda-Antikörper-Service, Berlin) or a monoclonal antibody against Gfp (Roche), goat anti-rabbit (Amersham™, GE Healthcare) and donkey anti-mouse (Pierce^®, Thermo Scientific) IgG peroxidase conjugate and Western Lightning Plus ECL solution (Perkin Elmer) were used. Densitometric quantification of proteins on blots was performed using ImageJ software.

Cultured EOCs were harvested with a cell lysis buffer (HEPES 10 nM pH 7.4, Na pyrophosphate 50 nM, NaF 50 nM, Na₂ EDTA 5 nM, EGTA 5 nM, Triton X-100 0.5%, Na₃VO₄ 2 nM, PMSF 1 nM, leupeptin 1 μg/ml, aprotinin 1 μg/ml). Protein levels were quantified by Bradford assay (Bio-Rad). The appropriate volume of SDS loading buffer (375 mM Tris-HCl pH 6.8, 6% SDS, 48% glycerol, 9% 2-mercaptoethanol, 0.03% bromophenol blue) was added to protein samples, heated for 5 min at 100 °C and loaded into 8% polyacrylamide gel. Proteins were transferred overnight to a PVDF membrane (Bio-Rad). The membrane was blocked for 1 hour at room temperature with blocking buffer (5% non-fat dry milk in TBS-T (50 mM Tris pH 7.6, 150 mM NaCl, Tween-20 0.05%)) and then incubated overnight with the appropriate primary antibodies. The membrane was washed 3 times with TBS-T and incubated with the appropriate HRP secondary antibodies (Santa Cruz goat anti-rabbit or goat anti-mouse). Next the membrane was incubated with a western lightning plus-ECL solution (Perkin Elmer). The chemiluminescence was measured using Chemidoc XRS + system (Bio-Rad) and quantified by densitometry using Quantity One software (Bio-Rad).

Total cell lysates were prepared in RIPA buffer containing protease and phosphatase inhibitors (Roche). Fifteen μg of protein were separated on 10–15% polyacrylamide SDS gels and electrotransferred onto PVDF membranes (Biorad) using wet tank sandwich method. Membranes were blocked and probed with the respective primary antibody: anti-YY1 (1:5000, rabbit monoclonal IgG EPR4652: ab109237; Abcam), anti-YY1 (1:5000, mouse monoclonal IgG₁ H10: sc7341; Santa Cruz Biotechnology), anti-YY1 (1:5000, rabbit polyclonal IgG H414: sc1703; Santa Cruz Biotechnology), anti-MYC (1:10000, rabbit monoclonal IgG Y69: ab32072; Abcam), anti-GAPDH (1:10000, mouse monoclonal IgG₁ 6C5: ab8245; Abcam), anti-histone H3 (1:1000, rabbit polyclonal IgG: #9715; Cell Signaling). Secondary antibodies were purchased from GE Healthcare Life Sciences and used at 1:10000 (anti-mouse IgG HRP-linked NXA931, anti-rabbit IgG HRP-linked NA934). Protein bands were detected by chemiluminescence (Western lightning Plus ECL Solution; Perkin Elmer) in the Advanced Fluorescence Imager machine (Intas) and protein signal quantification was performed by densitometry analysis using ImageJ software. Pearson’s correlation coefficient was determined by Microsoft Excel software (Microsoft Office Professional Plus 2010).