Ab16502

HUVECs were treated with apabetalone (5 or 20 μM) or DMSO and TNFα (10 ng/ml) for 2 h. Nuclear and cytoplasmic lysates were prepared using NE-PER kit (ThermoScientific, 78833) with freshly added protease inhibitor cocktail (BioShop or Sigma-Aldrich), phosSTOP phosphatase inhibitor (Roche 04906837001), and 0.5uM TSA (HDAC inhibitor). Protein concentration was determined with BioRad DC assay and lysate was added to NuPAGE LDS sample buffer (Novex/Invitrogen/Life Technologies NP0007) and 20 ug of total protein was loaded onto a NuPAGE 4-12% Bis-Tris gel (Novex/Invitrogen/Life Technologies NP0321BOX). For immunoblotting, the following primary antibodies were used: anti-p65 (Abcam, ab16502), anti-phospho p65 S536 (Cell Signaling, 3033), anti-BRD2 (Bethyl, A302-583A), anti-alpha tubulin (Sigma, SAB3500023), and anti-β-actin conjugated to peroxidase (Sigma, A3854). Secondary antibodies used were goat anti-rabbit IgG H&L chain specific peroxidase (Calbiochem, 401353) and rabbit anti-chicken IgY H&L chain specific peroxidase (Abcam, ab6753). Immunoreactive proteins were visualized by the chemiluminescent reagent ECLTM prime (GE Healthcare, RPN2232).

This analysis was performed to investigate changes in protein expression of regulatory factors implicated in cellular functions related to cell apoptosis and metastasis. Briefly, cultured chicken embryo fibroblasts (~1 × 10⁶ cells) were seeded and treated with GNR (A) and GNR (B) (~5.5 µg/mL) and supplemented with 10% fetal bovine serum (FBS) for 48 h to enhance the colloidal stability of the nanorods [70 (link)]. The cell lysate was then collected, and an equal number of proteins were resolved in 10% SDS PAGE gel, then transferred onto PVDF membranes. Empty binding sites of the membranes were blocked using 5% BSA. Membranes were blotted with the following primary antibodies: anti-JNK1, 2, 3 antibody (Abcam: ab225572), anti-NF-KB p65 antibody (Abcam: ab16502), anti-p38 MAPK antibody (Cell Signaling: 9212s), and anti-beta Actin antibody (Abcam: ab49900). The chemiluminescence was detected using ECL Western blotting substrate (Pierce Biotechnology, Pittsburgh, PA, USA) as described by the manufacturer, and blots were imaged using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The resulting bands were quantified using ImageJ software. Bands’ intensities normalized to β-actin were used to determine the relative protein expression.

The whole cell lysates of BV2 microglia or brain tissue samples (n = 3 mice per group) were extracted and collected by centrifugation (13,000 rpm, 10 min, 4°C). Protein concentrations were determined with Pierce™ BCA protein assay. Western blot analysis was performed as standard protocol. Protein samples were separated on 4–15% Tris-Gly SDS-PAGE gels (Beyotime, Shanghai, China) then transferred to PVDF membranes (Millipore, Bedford, United States). After blocking with TBST (5% non-fat dry milk or 5% BSA), the membranes were incubated with the primary antibody for phospho-IκBα (Ser32/36) (Cell signaling technology, 9246S, 1:1000), phospho-p65 (Abcam, ab16502, 0.5 μg/ml), NLRP3 (Cell signaling technology, 15101S, 1:1000), caspase-1 (Abcam, ab207802, 1:1000), TMS1/ASC (Abcam, ab151700, 1:5000), LC3B (Cell signaling technology, 83506S, 1:1000), SQSTM1/p62 (Abcam, ab109012, 1:10000), and Beclin1 (Abcam, ab62557, 1 μg/ml) at 4°C overnight. The membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. After thoroughly washed by TBST, membranes were incubated with the ECL western blotting substrate to detect the proteins. β-actin (Cell signaling technology, 3700S, 1:1000) served as loading control. ImageJ software was used to quantify the protein blots.