Rabbit anti phospho stat3 tyr705

Frozen liver tissue sections and cultured cells were fixed in 4% paraformaldehyde for 15 min, rinsed in 0.1% Tween 20 in phosphate-buffered saline, and incubated in blocking buffer (DAKO, Tokyo, Japan). The primary and secondary antibodies were diluted in 1% bovine serum albumin/phosphate-buffered saline and incubated with the cells for 1 h at 37°C. The slides were then mounted using DAPI, and the cells were viewed using an image analysis system (BIOREVO BZ-9000; KEYENCE, Osaka, Japan). The following primary antibodies were used: rabbit anti-LC3B, rabbit anti-ATG16L1, rabbit anti-phospho STAT3 Tyr705 (Cell Signaling Technology, Inc., Danvers, MA), and mouse anti-LAMP2 (Abcam, Cambridge, MA). The slides were then incubated with Alexa Fluor 488 (goat anti-rabbit) and Alexa Fluor 594 (goat anti-mouse)-conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Following treatment, microglial culture media were removed and cells were lysed with the 2x Laemmli sample buffer containing 5% 2-mercaptoethanol. The samples were then heated at 100°C for 5 min and vortexed before being loaded onto 4–15% precast acrylamide gels for electrophoresis. Gels were transblotted onto nitrocellulose membranes (0.45 µm), rinsed in TTBS (Tris-HCl with NaCl and Tween 20), and blocked with 5% Blotto (Santa Cruz, Dallas, TX) for 1 h at RT. Membranes were then probed with rabbit anti-β-actin (#4970) and rabbit anti-phospho-Stat3_(Tyr705) (#9131) or -Stat1_(Tyr701) (#9171) Abs (Cell Signaling Technology, Danvers, MA) overnight at 4°C. After washing 3x with TTBS, alkaline phosphatase (AP) conjugated secondary antibody (1 : 5,000 in 1% Blotto, Promega, Madison, WI) was added to the membranes at RT for 1 h. Following 3x washing with TTBS, membranes were rinsed in the assay buffer (1x) twice (2 min each) and then incubated in the substrate solution (CDP-Star, Applied Biosystems, Foster City, CA) for 10 min followed by imaging using an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).

In western blotting, equal amount of protein from the supernatant was separated using SDS–polyacrylamide gels in Tris/Glycine/SDS buffer, transferred onto PVDF membranes using Tris/Glycine buffer. Then, membranes were blocked with 5% nonfat milk in TBST and incubated with primary antibodies [rabbit anti- Phospho-STAT3 (Tyr705) from Cell Signaling Technology, 1:1000; rabbit anti-Ac-Lys685-STAT3 (acetylated STAT3) from Cell Signaling Technology, 1:1000; mouse anti-β-tubulin, 1:1000 from Cell Signaling Technology] overnight at 4˚C. After washing with TBST, the membranes were incubated with secondary antibodies (anti-rabbit-HRP, 1:3000 from Cell Signaling Technology; anti-mouse-HRP, 1:3000 from Cell Signaling Technology) at room temperature for 2 h. Protein bands were detected by ECL Western blotting detection reagents (GE Healthcare). The density of the target protein was quantified using ImageJ program. Then, target protein density was normalized to β-tubulin density.

Fifteen micrograms of total liver lysate protein or serum were loaded per well and were separated on 12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting and staining with rabbit anti-phospho-AKT (Ser 473) (1 : 1000, Cell Signaling Technology), rabbit anti-AKT (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-Stat3 (Tyr 705) (1 : 2000, Cell Signaling Technology), rabbit anti-STAT3 (1 : 2000, Cell Signaling Technology), orrabbit anti-caspase 3 (1,1000, Cell Signaling Technology). Signals were detected with Lumi-Light Western blot substrate (Thermo, Waltham, US) and exposure to X-ray film (GE Healthcare, Buckinghamshire, UK).

A detailed protocol can be found at protocols.io (DX.DOI.ORG/10.17504/PROTOCOLS.IO.HT4B6QW). Activation of the JAK-STAT3 neuroinflammation effector pathway [1 (link)] was assessed by quantifying pSTAT3^Tyr705 from immunoblots with detection of fluorescent signals using an infrared fluorescence scanner (Licor Biosciences; Lincoln, NE, USA) as described previously [1 (link),25 (link)–27 (link)]. Briefly, following incubation with primary antibodies (rabbit anti-phospho-STAT3^Tyr705[1:500]; Cell Signaling, Danvers, MA, USA), blots were washed with phosphate buffered saline with 0.1% Tween-20 and incubated with anti-rabbit fluorescent-labeled secondary antibody (1:2500) for 1h prior to scanning by Licor.

Proteins from samples (n = 4/group) were extracted in RIPA lysis buffer (Solarbio, China) with PMSF and a phosphatase inhibitor, and the protein concentration was assessed by BCA protein assay kit (Solarbio, China). Proteins were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide electrophoresis gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4°C in primary antibodies: rabbit anti-JAK2 (1:2,000; Cell Signaling Technology, United States), rabbit anti-phospho-JAK2 (Y1007 + 1,008) (1:2,000; Abcam, United States), rabbit anti-STAT3 (1:2,000; Cell Signaling Technology, United States), rabbit anti-phospho-STAT3 (Tyr705) (1:2,000; Cell Signaling Technology, United States), rabbit anti-β-actin (1:4,000; Proteintech Group, China). Later, the membranes were washed in TBST and then cultured with anti-rabbit HRP-conjugated secondary antibody (1:4,000; Proteintech Group, China) for 1 h. Finally, the enhanced chemiluminescence assay kit (Proteintech Group, China) was used to visualize immunoblots, and the densities of the relative target proteins were measured using ImageJ. The β-actin was chosen as the internal reference control.