Super signal ultra chemiluminescence detection reagents

Western blot analysis was performed as previously reported [24 (link)]. Monocytes and macrophage cells, after incubation of 24 and 48 h, were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer. The proteins were quantified by a bicinchoninic acid assay. Total protein extracts were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to nitrocellulose membranes. Blots were probed and incubated with the following primary antibodies: anti-iNOS (NOS2 (N-20), sc-651), anti-nitrotyrosine (PNK, sc-55256), anti-COX-2 (ab52237), and p-NF-kB (p65) (sc-166748). The Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL, USA) were used to detect the protein expression. A rabbit anti-human β-actin antibody (A5441; Sigma-Aldrich) was used as a control. A gel analysis software package (Gel Doc 1000; Bio-Rad) was employed to analyze the blot images. Results were expressed as mean values ± standard deviations (S.D.) of normalized densitometric values on the loading control.

Western blotting was performed as described below [29 (link)]. Briefly, total protein extracts were prepared by treating cells with lysis buffer (RIPA). In Western blot analysis, 50 μg of protein per lane was separated on a 4–12% NuPAGE gradient gel (Gibco Invitrogen, Paisley, UK). Blots were probed and incubated with rabbit polyclonal IgG anti-p38, anti-p-p38 (Thr 180/Tyr 182), anti-Erk1/2, anti-p-Erk1/2 (Thr202/Tyr204), anti-CXCL1, anti-CXCL5 and anti-CCL8 (Santa Cruz Biotechnology, Santa Cruz, CA) at 0.2 μg ml⁻¹ in Tris-buffered saline 0.1% Tween-20. A mouse anti-human monoclonal antibody recognizing the human β-actin (A5441; Sigma-Aldrich) was used as control in all experiments. Immunoblot signals were developed using Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL). The blot images were analysed with a gel analysis software package (Gel Doc 1000; Bio-Rad).

Western blot analysis was performed as reported previously [27 (link)]. U937 and HCT-116 cells were collected and lysed in RIPA buffer. Total protein extracts were separated on a 4–12% NuPAGE gradient gel (Gibco Invitrogen, Paisley, UK). Blots were probed and incubated overnight with primary antibodies for p-p38 (Tyr182, sc-101759), p-ERK (Thr202, sc-101760), and β-actin (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL, USA) were used to detect the protein expression. A gel analysis software package (Gel Doc 1000; Bio-Rad) was employed to analyze the blot images.