Mouse β actin

As previously described by us,
⁴⁰ (link) proteins were extracted from the MCA‐supplied brain regions and loaded onto SDS‐polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti‐NLRP3, Merck; 1:500, rabbit anti‐ASC, Abcam; 1:1000, rabbit anti‐IL‐1β, Abcam; 1:1000, rabbit anti‐IL‐18, Proteintech; 1:1000, rabbit anti‐caspase‐1, Abcam; 1:500, rabbit anti‐GSDMD, Affinity; 1:2000, rabbit anti‐G3BP1, Proteintech; 1:500, rabbit anti‐TIA1, Proteintech; 1:1000, rabbit anti‐DDX3X, Proteintech; 1:1000, rabbit anti‐IgG antibody, Cell Signaling Technology; 1:10,000, rabbit anti‐SYN, Abcam; 1:1000, rabbit anti‐PSD‐95, Cell Signaling Technology; 1:1000, rabbit anti‐BDNF, Abcam; 1:500, rabbit anti‐Ang‐1, Proteintech; 1:500, rabbit anti‐Ang‐2, Proteintech; 1:500, rabbit anti‐VEGF, Abcam; 1:1000, mouse β‐actin, Santa Cruz) for 24 h at 4°C. Next, membranes were incubated with a secondary antibody (1:4000, goat anti‐rabbit IgG, goat anti‐mouse IgG, Cell Signaling Technology) for 2 h at room temperature. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health) to quantify protein expression in terms of relative image density.

Hippocampal tissues were homogenized on ice and lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 1 mM PMSF, and leupeptin 100 mg/mL. The protein content was measured using a Colorimetric Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of protein was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse β-actin (1:1000; Santa Cruz Biotechnology, CA, USA), Bax (1:1000; Cell Signaling, Danvers, MA, USA), Bcl-2 (1:1000; Santa Cruz Biotechnology, CA, USA), BDNF (1:1000; Alomone, Jerusalem, Israel), PSD95, 1:1000; Cell Signaling, Danvers, MA, USA), primary antibodies. Horseradish peroxidase-conjugated secondary anti-mouse antibodies were used for β-actin and Bcl-2; anti-rabbit conjugated secondary antibodies were used for, Bax, BDNF, and PSD95.

As described previously (Wang et al., 2017 (link)), the freshly colonic tissues were excised and fragmented using a scalpel. 500 ug–1 mg tissue portions were lysed for 30 min on ice with RIPA lysis buffer (Beyotime) in the presence of cocktail protease inhibitor (Roche) and phoSTOP phosphatase inhibitor (Roche). An additional step of sonication was also performed. Protein extracts (35 µg) were boiled and subjected to 10% SDS-PAGE gel before transfer to nitrocellulose membranes. The membranes were blocked for 2 h in PBST (PBS with 0.5% Tween 20) with 5% non-fat dry milk (Bio-Rad) and incubated with specific primary antibodies at 4°C overnight. Appropriate HRP-conjugated second antibodies (Cell Signaling Technology, #7076) were used at 1:3,000 dilution for 2 h at room temperature. Signals were detected by ECL HRP substrate (Advansta). The following primary antibodies were used: mouseβ-actin (Santa Cruz, sc-47778), mouse NF-κB p65 (Cell Signaling Technology, #6956), and mouse TNF-α (Abcam, ab1793).

Hippocampal tissues were homogenized on ice and lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 1 mM PMSF, and leupeptin 100 mg/mL. The protein content was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA). Thirty micrograms of protein was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse β-actin (1:1000; Santa Cruz Biotechnology), GAPDH (1:3000; Santa Cruz Biotechnology), t-Akt and p-Akt (1:1000; Cell Signaling), t-GSK3β and p-GSK3β (ser 9) (1:1000; Cell Signaling), t-Tau and p-Tau (ser202/Thr205 (1:1000; Thermo Fisher), Bcl-2 and cytochrome C (1:1000; Santa Cruz Biotechnology), Bax (1:1000; Cell Signaling), cleaved caspase-3 (1:700; Cell Signaling), brain-derived neurotrophic factor (BDNF; 1:1000; Alomone), PSD95 (1:1000; Cell Signaling), and synaptophysin (1:1000; Abcam) primary antibodies. Horseradish peroxidase-conjugated secondary anti-mouse antibodies were used for Bcl-2, p-Tau, cytochrome C, β-actin, and GAPDH; anti-rabbit conjugated secondary antibodies were used for t-Akt, p-Akt, t-tau, p-tau, t-GSK3β, Bax, cleaved caspase-3, BDNF, PSD95, and synaptophysin.

The cells were lysed for western blot analysis using lysis buffer, and the western blot was performed as described previously.^{10 (link),14 (link)} Immunoblot analyses were performed with mouse anti-peroxisome proliferator-activated receptor (PPAR) γ (#sc-7273, 1:1000), rabbit anti-CCAAT-enhancer-binding proteins (C/EBP)α (#sc-61, 1:500), rabbit anti-C/EBPβ (#sc-150, 1: 2000), rabbit anti-heme oxygenase (HO)-1 (#sc-10789, 1:1000), and mouse β-actin (#sc-47778, 1:10000) that were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit phospho-NF-κB p65 (Ser536) (#3034, 1:1000) and rabbit NF-κB p65 (D14E12) (#8242, 1:1000) were purchased from Cell Signaling Technology, Inc. (Boston, MA). Immunoreactive bands were detected by means of the Enhanced Chemiluminescence Detection Kit (Amersham Pharmacia Biotech), and X-ray film was exposed to the bands (Amersham Pharmacia Biotech). The signals were quantified by densitometry and normalized to the β-actin level in the same sample.