Rabbit anti akt

Western blotting was performed as previously described [29 (link)], using the following antibodies: mouse anti-Cbl-b, mouse anti-c-Src, and rabbit anti-β-actin antibodies were obtained from Santa Cruz Biotechnology; rabbit anti-Akt, anti-p-Akt (Ser473), anti-ERK1/2, anti-p-ERK1/2 (Thr202/Tyr204) and anti-p-Src (Y416) antibodies were obtained from Cell Signaling Technology, mouse anti-RANK antibodies were obtained from R&D, USA. Proteins were visualized using the enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, Rockford, IL, USA) and signals were quantitated using NIH Image J software.

Protein samples were homogenised in lysis buffer (20 mM Tris, 150 mM NaCl, 1% (v/v) Triton X-100) containing 1 mM PMSF, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, and leupeptin. The samples were then incubated for 30 min at 4 °C and centrifuged for 10 min at 13,000 × g. The supernatant (lysate) was collected, and 15 μg protein was loaded into each lane. Samples were separated on 8–12% SDS-polyacrylamide gels and electro-blotted onto nitrocellulose membranes (Millipore). After blocking with 5% (w/v) non-fat milk, the blots were incubated with the following primary antibodies: rabbit anti-phospho-Akt (p-Akt-Ser473, 1:400, Santa Cruz), rabbit anti-Akt (1:400, Santa Cruz), rabbit anti-phospho-GSK-3β (p-GSK-3β-Ser9, 1:1000, Cell Signaling Technology), rabbit anti-GSK-3β (1:1000, Cell Signaling Technology), mouse anti-phospho-tau (p-tau-Ser396, 1:1000, Cell Signaling Technology), mouse anti-tau (1:1000, Cell Signaling Technology), rabbit anti-presenilin 1 (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz). Conjugated goat anti-rabbit or goat anti-mouse IgG was detected with enhanced chemiluminescence (ECL) (Pierce^® ECL Western Blotting Substrate). β-actin was used as an internal reference for relative quantification.

The following primary antibodies were used for western blotting (WB) and immunocytochemistry (ICC): rat anti-mouse CD11b (1:400 for ICC, Abcam), rabbit anti-COX-2 (1:200 for ICC, Abcam), rabbit anti-IL-1β (1:200 for ICC, Abcam), rabbit anti-GFAP (1:500 for ICC, Wako, Japan), rabbit anti-Iba-1 (1:500 for ICC, Wako), goat anti-Iba-1 (1:500 for ICC, Wako), rabbit anti-AKT (1:1000 for WB, Santa Cruz Biotechnology), rabbit anti-p-AKT (Ser473) (1:1000 for WB, Cell Signaling Technology), rabbit anti-ERK (1:1000 for WB, Santa Cruz Biotechnology), rabbit anti-p-ERK (Thr42/44) (1:1000 for WB, Cell Signaling Technology), rabbit anti-STAT3 (1:1000 for WB, Cell Signaling Technology), rabbit anti-p-STAT3 (Ser727, 1:1000 for WB, 1:200 for ICC, Abcam), rabbit anti-JNK (1:1000 for WB, MyBioSource, San Diego, CA, USA), rabbit anti-p-JNK (Thr183/Tyr185, 1:1000 for WB, MyBioSource), rabbit anti-P38 (1:1000 for WB, Cell Signaling Technology), rabbit anti-p-P38 (1:1000 for WB, Cell Signaling Technology), rabbit anti-TLR4 (1:1000 for WB, Thermo Scientific, Waltham, MA, USA), and rabbit anti-TLR4 (1:1000 for WB, Novus Biologicals, Littleton, CO, USA). We used a TLR4 inhibitor (TAK-242, 500 nM, Calbiochem), AKT inhibitor (MK2206, 10 μM, Selleckchem), and STAT3 inhibitor (S3I-201, Sigma-Aldrich) in our experiments. LPS from Escherichia coli O111:B4 was purchased from Sigma-Aldrich (St. Louis, MO, USA).