Ab229381

A 1 cm nitrocellulose membrane strip was spotted with spinal cord homogenates (30 µg protein), dried, blocked (5% BSA/TBS-T, 1 h, RT) and incubated (30 min, RT) with the following antibodies: anti-IL-1β (1:1000), anti-TNF-α (1:1000), anti-IL6 (1:500, ab229381, abcam) or anti-β actin (1:2500, A5441, Sigma). Then washed 3x (TBST-T) and incubated with HRP-conjugated secondary antibodies (1:2000, 30 min, RT)^{50 (link)}. BSA and recombinant cytokines were used as negative and positive controls, respectively. Signals were developed (ECL, Thermo Fisher, 34577) and read (iBright FL1500 Imaging System). Integrated density was measured using FIJI.

The vaginal tissues were lysed and homogenized in RIPA buffer with phenylmethylsulfonyl fluoride (PMSF) and quantified with a BCA kit (Keygen Biotech, KGP902, China). The protein samples (30 μg/lane) were separated using SDS-PAGE (Beyotim, P0012A, China) and transferred to polyvinylidene difluoride membranes (PVDF) (Merck, IPVH00010, United States) after electrophoresis. The membranes were blocked with 5% skimmed milk for 3 h, followed by incubation with TNF-α (1:1,000, Abcam, ab205587, United States), IL-1β (1:1,000, Abcam, ab234437, United States), IL-6 (1:1,000, Abcam, ab229381, United States), Dectin-1 (1:1,000, Abcam, ab140039, United States), Syk (1:1,000, Cell Signaling Technology, 13198T, United States), PLCγ-2 (1:1,000, Cell Signaling Technology, 3872T, United States), CARD9 (1:500, Affinity, DF8387, China), NF-κB (1:1,000, Cell Signaling Technology, 8242T, USA) and β-actin (1:2,000, Affinity, AF7018, China) antibodies overnight at 4°C. Then, the membranes were incubated with an HRP-conjugated antibody (1:5,000, Affinity, S0001, China) at room temperature for 1 h. The membranes were visualized with a chemiluminescence (ECL) kit by a Tanon 5200 system (Shanghai, China). ImageJ software (Version 1.52a) was used to measure the protein bands based on that of β-actin.

Western blot was performed as described previously [28 (link)]. The primary antibodies were as follows: anti-IGFBP1 (ab180948, Abcam), anti-Rev-erbα (sc-393215, Santa Cruze), anti-β-Actin (ab179467, Abcam), anti-β-Tubulin (ab179513, Abcam), anti-PR (human, 8757, Cell Signaling Technology), anti-C/EBPβ (ab32358, Abcam); anti-IL-6 (human, ab233706, Abcam), anti-IL-6R (human, ab222101, Abcam), anti-PR (mouse, ab133526, Abcam), anti-IL-6 (mouse, ab229381, Abcam), anti-IL-6R (mouse, ab300581, Abcam), anti-Wnt4 (sc-376279, Santa Cruze). β-Tubulin and β-Actin were used as internal standards.