Anti phospho erk1 2 polyclonal antibody

Overnight serum-starved LβT2 cells were treated with 100 μM palmitate for 0.5, 1, 2, 5, 10, or 15 min and then lysed in an extraction buffer (50 mM Tris-Cl, 150 mM NaCl, 1 mM
EDTA, and 1% Triton X-100) containing 1% protease inhibitors (Nakarai Tesque, Kyoto, Japan) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total cell lysates were then
centrifuged at 15,000 g for 5 min. Supernatants were mixed with 4 × sodium dodecyl sulfate sample buffer, boiled, and then separated in polyacrylamide gels. The
proteins were then transferred on to polyvinylidene difluoride membranes (Millipore, San Jose, CA, USA). The membranes were probed with anti-ERK1/2 polyclonal antibody (1:1,000;
Cell Signaling Technology, Danvers, MA, USA) or anti-phospho-ERK1/2 polyclonal antibody (1:1,000; Cell Signaling Technology). The membranes were further incubated with anti-rabbit
IgG antibody conjugated to horseradish peroxidase (HRP) (1:4,000, Cell Signaling Technology), and developed with ImmunoStar Zeta (Fujifilm Wako Chemicals). Chemiluminescence was
recorded using the ImageQuant LAS 500 spectrometer (GE Healthcare, Chicago, IL, USA).

Given the limited number of microglia in primary culture available for protein extraction, the in-cell western blot assay was performed as previously described (Egorina et al., 2006). Briefly, cells were seeded in 96-well plates (Black wall, Corning 3631) at 5 × 10⁵ cells/well and cultured for 12 hours. Cell were washed twice in PBS, fixed with 4% paraformaldehyde for 20 minutes, incubated with 0.1% Triton X-100 for 20 minutes, and then washed twice in PBS. Blocking was carried out with 5% bovine serum albumin for 1 hour at room temperature. The cells in the wells were incubated overnight at 4°C with the negative control or a mixture of anti-phospho-ERK1/2 polyclonal antibody (1:500; Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin monoclonal antibody (1:1,500; Santa Cruz Biotechnology), and then with secondary antibodies (Alexa Fluor 800-conjugated goat anti-mouse IgG and/or IRDye 800-conjugated goat anti-rabbit IgG, 1:5,000; Li-COR Biosciences Inc., Lincoln, NE, USA) at room temperature for 1 hour. The immunoreactivity of cells was quantitatively analyzed using an Odyssey IR imaging system (Li-COR Biosciences Inc.) and expressed as the mean optical density. The amount of each protein was normalized to that of β-actin as follows: optical density of target protein/optical density of the corresponding β-actin.