Anti gadph antibody

Western blot analysis was performed as previously described [30 (link)]. The antibodies used in this study included an anti-CD86 antibody (1:200; ab220188; Abcam, MA, USA), anti-NF-κB p65 antibody (1:200; ab32536; Abcam, MA, USA), anti-GADPH antibody (Santa Cruz Biotechnology, CA, USA), anti-Histone H3 (1:1000; ab1791; Abcam, MA, USA), and goat anti-Rabbit IgG H&L (HRP; 1:2000; ab6721; Abcam, MA, USA). The original image of the WB stain is given in Additional file 5.

Cells were harvested under the described conditions and lysed in a RIPA buffer (0.5% Non-idet P-40, 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄) with a protease inhibitor (1 mM PMSF). The BCA protein assay was used to quantify protein. Briefly, after Mix reagents, sample were added and incubated for 30 min and read at 562 nm. Equal quantities of protein were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following transfer to PVDF membranes, samples were incubated with primary antibody (1:1000 dilution) at 37°C for 1 h or overnight at 4°C. The membrane was washed twice with TBS, and the bound antibody was detected using an IgG-HRP secondary antibody (1:500 dilution) for 30 min. Blots were detected using an enhanced chemiluminescence system (Amersham, UK). Another membrane prepared by the same protocol was probed with anti-GADPH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) to normalize sample loading.

Cultured MNK-45 and SGC 7901 cells were respectively homogenized in cell lysis solution (Sigma) and centrifuged at 12,000 xg for 30 min at 4°C. Supernatants were collected after centrifugation, and protein concentrations were determined using a BCA Protein Assay kit (Pierce). In total, 30 μg of protein was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene membrane and treatment with a rabbit anti-human PADI4 antibody (Sigma). This antibody targets amino acids 91-107 of human PADI4. Western blotting performed by manufacturer demonstrated that this antibody has no cross-reactivity among other PAD family members. The polyvinylidene membranes were subsequently rinsed with a wash solution and incubated with sheep anti-rabbit IgG conjugated to peroxidase (Beyotime Biotech) for 1 h. Following another wash step, the signal was detected using an enhanced chemiluminescence (ECL) plus kit (Beyotime Biotech). Another membrane was prepared using the same protocol and probed with an anti-GADPH antibody (Santa Cruz) to normalize sample loading.
The expression levels of CXCR2, KRT14, TNF-α and the His tag in the cultured cells were examined by the same western protocol described above. Commercial antibodies against these proteins were obtained from CST, Sigma, Abcam and Abcam, respectively.