Anti erk1 2 clone 137f5

Macrophages (10⁶ per condition) were treated as indicated, washed once with ice cold PBS, and lysed with RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP–40, 0.5% sodium deoxycholate, 0.1% SDS) with addition of a protease and protein phosphatase inhibitor cocktail (Pierce, Rockford, IL). Lineage-depleted bone marrow cells and FACS-isolated bone marrow monocytes and granulocytes were lysed as above. Samples were boiled in reducing sample buffer (Bio-Rad) and run on SDS-PAGE gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and blocked in 5% nonfat dry milk in PBS–0.1% Tween 20. Primary antibodies included anti-phospho-ERK1/2 (clone D13.14.4E), anti-ERK1/2 (clone 137F5), and anti-β-actin (clone D6A8) (all from Cell Signaling Technology, Danvers, MA). Primary antibodies were incubated in blocking buffer overnight at 4°C; membranes were then washed three times in PBS–0.1% Tween 20, treated with secondary antibody (horseradish peroxidase-linked goat anti-rabbit IgG, catalog number 7074, Cell Signaling Technology) in blocking buffer, washed three times, and treated with enhanced chemiluminescence reagent (Pierce). Membranes were exposed to film (Amersham GE Healthcare, Pittsburgh, PA), and images were scanned and analyzed. Densitometric analysis was performed using ImageJ software [17 (link)].

The following mouse monoclonal antibodies were used as primary antibodies: anti-phospho-ERK1/2 (Thr202/Tyr204) (clone E10, #9106; Cell Signaling Technologies, MA, USA), anti-β-actin (clone AC-15, #A5441; Sigma-Aldrich Corporation, MO, USA), anti-desmoplakin (clone 11-5F; gift from David Garrod, University of Manchester, UK), anti-keratin 14 (clone LL001; Purkis et al., 1990 (link)), anti-keratin 10 (clone DE-K10, #M7002; Agilent Technologies, CA, USA), anti-E-cadherin (clone 36, #610182; BD Biosciences, CA, USA) and anti-Ki-67 (clone MM1, #NCL-L-Ki67-MM1; Leica Biosystems, IL, USA). The following rabbit monoclonal or polyclonal antibodies were also used as primary antibodies: anti-phospho-AKT (Ser473) (clone D9E, #4060), anti-AKT (clone C67E7, #4691), anti-phospho-ERK1/2 (Thr202/Tyr204) (clone D13.14.4E, #4370), anti-ERK1/2 (clone 137F5, #4695) and anti-YAP (clone D8H1X, #14074) were purchased from Cell Signaling Technologies (MA, USA). Secondary antibodies used were as follows: goat anti-rabbit IgG (H+L) Alexa Fluor 594 (#A-11037) and goat anti-mouse IgG (H+L) Alexa Fluor 568 (#A-11031) were purchased from Thermo Fisher Scientific (MA, USA), anti-mouse IgG (H+L), HRP conjugate (#W4021) and anti-rabbit IgG (H+L), HRP conjugate (#W4011) were purchased from Promega Corporation (WI, USA).