Ab178876

Ovarian cancer cells were disrupted using cell lysis buffer (Promega) on ice for 30 min and centrifuged at 12000 rpm for 30 min. The supernatant was transferred into the new centrifuge tube, and the concentration of protein samples was detected using the BCA-200 Protein Assay kit (Pierce, Rockford, IL, USA). Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore). The non-specific sites in the membrane were blocked using 5% skim milk for 1 h, followed by incubation with primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibody. The blots were visualized using the enhanced chemiluminescent (ECL) system (Beyotime, Shanghai, China). The primary antibodies, including phosphorylated extracellular regulated MAP kinase (p-ERK; ab214036), ERK (ab17942), p-MAP kinse-ERK kinase (p-MEK; ab96379), MEK (ab178876) and GAPDH (ab181602) were purchased from Abcam (Cambridge, MA, USA).

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.

Cells in experimental and control groups were lyzed and centrifuged. After determined the protein concentrations, the cell lysate samples (30 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto NC membranes. The membranes were blocked with 5% BSA in TBST and immunoblotted by the primary antibodies. The primary antibodies used in this study were:anti-GALNT6 (1:1000, sc-100755, Santa Cruz Biotechnology), anti-E-cadherin (1:500, 13-1700, Invitrogen), anti-N-cadherin (1:200, sc-393933, Santa Cruz Biotechnology), anti-Slug (1:500, ab27568, Abcam), anti-GRP78 (1:1000, ab21685, Abcam), anti-phospho-MEK1/2 (1:1000, #9154, CST), anti-MEK1/2 (1:1000, ab178876, Abcam), anti-phospho-ERK1/2 (1:1000, #4370, CST), anti-ERK1/2 (1:1000, #4695, CST), biotinylated anti-Viciavillosa agglutinin (specific to GalNAc-Ser/Thr) (VVA, 1:1000, B-1235, Vector) and anti-GAPDH (1:2000, 10494-1-AP, Proteintech). The secondary antibodies were HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:5000, SA00001-1, Proteintech), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:5000, SA00001-2, Proteintech) and HRP-streptavidin (1:10000, SA-5014, Vector). The protein bands were visualized by enhanced chemiluminescent reagents (Advansta) and the intensity of protein band was quantified by ImageJ software.