Cell lysis solution

Cell lysates were homogenized in Cell Lysis Solution (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged for 5 min at 4°C. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 3 h and then transferred to polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ, USA) for an additional 2 h. Antibodies against human PADI2 (Proteintech, Rosemont, IL, USA; 2,000-fold dilution) were used for Western blot analysis. After incubation with specific antibodies against β-actin (Sigma-Aldrich) at 4°C overnight, the membranes were washed with 1% Tris-buffered saline containing Tween 20 in triplicate, incubated with the appropriate secondary antibodies for 1 h and detected by chemiluminescence.

The cells were collected using cell lysissolution (Sigma) and denatured at 100 °C for 5 min. The protein concentrations were tested using bicinchoninic acid (BCA) Protein Assay Kit (Pierce, USA). Dodecyl sulphate polyacrylamide gel (SDS-PAGE) electrophoresis was performed to separate the proteins. Next, the proteins were transblotted onto nitrocellulose membranes (Amersham, USA). Primary antibodies were added after the membranes have been blocked with 5% not-fat milk. Then, the membranes were maintained at 4 °C overnight. Primary antibodies were as follows: anti-Col2A1 (1:8000, ab34712,abcam), anti-E2F2 (1:1000, ab65222), anti-p16INK4a (1:5000,ab108349) and anti-β-actin (ab8226,1:5000). Secondary antibodies (abcam) were incubated at room temperature for 2 h. The blot bands were developed with Enhanced chemiluminescence (Amersham).The density of bands was quantified using Quantity one 4.6.2.

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.