Anti erk 137e5

Cells (1 × 10⁵) were washed once with PBS (without calcium), lysed in 100 μl of SDS-loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 2% ß-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and then subjected to SDS-PAGE. The separated proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), which was then incubated with 1: 1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology) as recommended by the manufacturers. Primary antibody binding was detected using a Clarity Western ECL substrate (Bio Rad, Hercules, CA), and images were captured with a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) (20G11, Cell Signaling Technologies, Danvers, MA, USA), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (T308) and anti-Akt pan (C31E5E and C67E7, Cell Signaling), anti-phospho-NF-κB p65 (S536) and anti-NF-κB p65 (93H1 and D14E12, Cell Signaling), anti-IκBα (L35A5, Cell Signaling), anti-pIκBα (Ser32, 14D4, Cell Signaling) and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).

Cells (1×10⁵) were washed once with PBS, lysed in 100 μl SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, and 2% β-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and subjected to SDS-PAGE, and the separated proteins were transferred onto a PVDF membrane (Merck Millipore) that had been incubated with 1:1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling), as recommended by the manufacturers. Primary antibody binding was detected using a Clarity™ Western ECL substrate (Bio Rad, Hercules, CA, USA), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-pERK (20G11, Cell Signaling), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (Thr308) (C31E5E, Cell Signaling), anti-Akt pan (C67E7, Cell Signaling), anti-phospho-FAK (Tyr925) (Cell Signaling), anti-FAK (Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), anti-NF-κB p65 (D14E12, Cell Signaling), and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).

An anti-human APC-CD271 antibody (clone ME20.4-1.H4, Miltenyi Biotec, Germany) was used for flow cytometry analysis. The following antibodies were used for Western blotting analysis: anti-phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) (20G11, Cell Signaling Technologies, Danvers, MA, USA), anti-ERK (137E5, Cell Signaling), anti-phospho-NF-κB p65 (S536) and anti-NF-κB p65 (93H1 and D14E12, Cell Signaling), anti-phospho-Iκbα (Ser32) and anti-Iκbα (14D4 and L35A5, Cell Signaling), anti-phospho- Akt (S473) and anti-Akt (pan, Cell Signaling) anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA), anti-involucrin (clone SY-5, Sigma-Aldrich, St. Louis, IL, USA), anti-CDKN1C (NBP1-89917, Novus Biologicals, Littleton, CO, USA), and anti-β-actin (AC-15, Sigma-Aldrich). The following antibodies were used for immunohistochemistry analysis: anti-CD271 (C40-1457, BD Biosciences, Franklin Lakes, NJ, USA), anti-Ki67 [clone 30-9 (Roche, Switzerland) for DAB staining and clone SP6 (Abcam, UK) for immunofluorescence staining] and anti-involucrin (clone SY-5, Sigma-Aldrich).