Ab263947

Samples were homogenized in lysis buffer (pH 7.4) containing a protease inhibitor cocktail (HY-K0010, MedChenExpress), phosphatase inhibitor cocktail III (HY-K0023, MedChenExpress), 50 mM Tris, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% NP-40 (Sigma-Aldrich). The protein concentration was determined using a BCA Protein Assay kit (Tiangen Biotech). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the samples were blotted onto a polyvinylidene fluoride (PVDF) membrane. Antibodies against DPP4 (ab28340, Abcam), F4/80 (28463-1-AP, Proteintech), CD206 (18704-1-AP, Proteintech), p44/42 MAPK (Erk1/2) (137F5) rabbit mAb (4695, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, Cell Signaling Technology), signal transducer and activator of transcription 6 (STAT6) (ab32520, Abcam), and Phospho-STAT6 (phospho Y641) (ab263947, Abcam) were used at a dilution of 1:2000. Secondary antibody binding and detection were performed according to standard protocols using an enhanced chemiluminescence (ECL) detection reagent (Bio-Rad).

Western blot analysis was performed following the protocol from previous studies (Fan Y. et al., 2015 (link)). The anesthetized rats were transcardially perfused with saline. The brains were removed, and the hippocampi were dissected. Lysis buffer was prepared by RIPA and PMSF according to a ratio of 100:1, and one tablet phosphatase inhibitor (4906837001, Roche, Switzerland) was added to every 10 mL lysis buffer. The tissues were homogenized in lysis buffer (containing phosphatase inhibitor). After cracking and centrifugation, the supernatant was collected. Equal amounts of total proteins were resolved by SDS-PAGE and transferred on to a nitrocellulose membrane. The membranes were incubated overnight at 4°C with the following primary antibodies: anti-STAT6 (ab217998, 1:1,000, Abcam, USA), anti-pSTAT6 (ab263947, 1:1,000, Abcam, USA), anti-TGF-β1 (ab92486, 1:1,000, Abcam, USA), anti-IL-10 (DF6894, 1:500; Affinity, USA), anti-IL-1β (AF5103, 1:500; Affinity, USA), anti-IFN-γ (ab9657, 1:1,000, Abcam, USA), and anti-α-tubulin (2144, 1:1,000, CST, USA). Then, the membranes were washed with 1 × TBST (10 mM Tris-HCl+10 mM NaCl+0.05%Tween-20), incubated with appropriate secondary antibodies: HRP-conjugated anti-rabbit antibody (5571, 1:3,000, CST, USA), at room temperature for 1 h, and then treated with ECL substrate (Millipore, USA).

Proteins were collected, quantified with a BCA kit, and boiled samples (20 μg) were separated via SDS-PAGE, then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h and then incubated with primary antibodies, including anti-p-STAT6 (ab263947, Abcam, Cambridge, UK, 1:1000), anti-STAT6 (ab32520, Abcam, 1:1000), anti-p-JAK2 (ab32101, Abcam, 1:1000), anti-JAK2 (ab108596, Abcam, 1:1000), anti-p-STAT3 (ab76315, Abcam, 1:1000), anti-STAT3 (ab68153, Abcam, 1:1000), anti-Bcl-2 (ab32124, Abcam, 1:1000) and anti-β-tubulin (TA503129, OriGene, Rockville, MD, USA, 1:1000) at 4 ℃ overnight. Next, the membrane was incubated with an HRP-conjugated secondary antibodies for 1 h and proteins were visualized using the ECL Western Blotting Detection System (ChemiDoc XRS, Bio-Rad, Hercules, CA, USA).

After lysis of mouse retinas in the RIPA buffer (Beyotime, Shanghai, China), the BCA approach (Beyotime) was utilised to determine protein content. Proteins (around 20 μg) were separated through 10% or 12% SDS-PAGE, followed by transfer on PVDF membranes (0.45 μm; Millipore, Shanghai, China). Later, 5% Bovine Serum Albumin was utilised to block membranes under ambient air for a 1-h period, followed by overnight incubation under 4°C using anti-Iba1 (1:1000, #ab178846, Abcam, Cambridge, MA, USA), anti-iNOS (1:1000, #ab178945, Abcam, Cambridge, MA, USA), anti-CD206 (1:200, AF2535, R&D Systems), anti-STAT1 (1:1000, #ab239360, Abcam, Cambridge, MA, USA), anti-STAT1 (phospho S727, 1:1000, #ab109461, Abcam, Cambridge, MA, USA), anti-STAT6 (1:1000, #ab32520, Abcam, Cambridge, MA, USA), and anti-STAT6 (phospho Y641, 1:1000, #ab263947, Abcam, Cambridge, MA, USA), with β-Tubulin being the endogenous reference. Membranes were rinsed sufficiently through PBST three times. Subsequently, the membrane was exposed to 1-h HRP-labelled secondary antibody incubation. At last, chemiluminescence was used to analyse the expression of each protein.