Immunoblot Analysis of Microglia and Brain

Immunoblot analysis was performed as previously described^{29 (link)} with the following sample preparation specification. Primary microglia were washed in 4 °C DPBS and then incubated in Pierce RIPA lysis buffer supplemented with 1× Halt Protease and phosphatase inhibitors (complete RIPA; Thermo Fisher Scientific) for 15 min at 4 °C. For brain samples, cortices were dissected from PBS-perfused 12-month-old male and female mice. Tissues were incubated in complete RIPA for 30 min at 4 °C. Primary antibodies were: p-NCF2 (1:1000, rabbit polyclonal, PA5-105094, Thermo Fisher Scientific); NCF2 (1:1000, rabbit polyclonal, PA5-37323, Thermo Fisher Scientific), p-PXN (1:1000, rabbit polyclonal, PAB7932, Abnova), paxillin (1:1000, rabbit monoclonal, ab32115, Abcam), p-MEK2 (1:1000, rabbit polyclonal, 28955-1-AP, Thermo Fisher Scientific), MEK1/2 (1:10,000, rabbit monoclonal, ab178876, Abcam) and GAPDH (1:10,000, rabbit monoclonal, 2118, Cell Signaling Technology). Primary antibodies were visualized with horseradish peroxidase-conjugated secondaries (Cell Signaling Technology) and ECL reagents. Densitometry was performed using NIH ImageJ (v.1.50), with protein values for each band normalized to GAPDH from the same membrane.