Anti human β actin

BxPC3 cells were stimulated with different concentrations of S100P (0.03–3 μM) for 60 min To test the inhibitory effect of the anti-S100P mAbs, 2 μM of each was incubated for 2 h with S100P protein (1 μM) at 37 °C, prior to the addition to the cells. SDS–PAGE and western blot analyses were performed as described previously.^{20 (link)}The following antibodies were used: 4B12 mouse mAb anti-human S100P (Leitat Technological Center, Barcelona, Spain) at 1 μg/ml; peroxidase-conjugated mAb anti-human β-Actin (Sigma) at a 1:25 000 dilution; mouse mAb anti-human phospho-IκB-α (Ser32/36) (Cell Signaling Technology, Danvers, MA, USA), at a 1:1000 dilution; rabbit polyclonal antibody anti-human IκB-α (Cell Signaling Technology), at a 1:1000 dilution. Goat anti-mouse (Jackson ImmunoResearch, Baltimore, PA, USA) at 0.04 μg/ml and goat anti-rabbit (Sigma) at a 1:25 000 dilution, were used as secondary antibodies.
Bands were quantified using the NIH ImageJ imaging software. Quantification of the protein expression was performed by densitometric analysis referring the results to the control cells in the non-stimulated condition (that represents 100% of expression). All signals intensities were normalized to β-Actin.

Primary tumors and mesenteric metastatic lesions were collected and homogenized in PBS with protease inhibitors (Roche, Mannheim, Germany) with a QIAGEN TissueRuptor (Qiagen). Homogenates were sonicated on ice for 30 seconds and then spun down at 4 °C at maximum speed in a table top centrifuge for 20 minutes. Samples in reducing Laemmli buffer were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis and blots were incubated with primary anti-human DSG2 (AbD Serotec, Raleigh, NC, 6D8, 1:1,000) and anti-human β-actin (Sigma) (1:3,000) antibodies and a horse radish peroxidase (HRP)-linked secondary anti-mouse IgG antibody (1:2,000).