Ab7090

The A549/DDP cell line or tumor tissues were rinsed with PBS and lysed in a RIPA buffer with protease inhibitors and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA) for Western blots. The proteins were segregated from tumor samples or cell lines. Procedures for immunoblotting are described elsewhere [18 (link)]. Primary antibodies against IL-7 (Abcam, ab9732, 1 : 3000 dilution), IL-7R (Abcam, ab180521, 1 : 1000 dilution), PI3K (CST, 13666, 1 : 1000 dilution), p-PI3K p85 alpha (phospho Y607) (Abcam, ab182651, 1 : 1000 dilution), AKT (CST, 4691, 1 : 1000 dilution), p-AKT (CST, 4691, 1 : 2000 dilution), ABCG2 (Abcam, ab130244, 1 : 1000 dilution), and GAPDH (Abcam, ab181602, 1 : 10000 dilution), as well as HRP-conjugated goat anti-rabbit secondary antibodies (Abcam, ab7090, 1 : 10000 dilution) and HRP-conjugated goat anti-mouse secondary antibodies (Abcam, ab47827, 1 : 5000 dilution), were procured from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Denver, MA).

We utilized cell lysis buffer (Beyotime), containing 1% PMSF (Amresco), to cleave proteins for 30 minutes on ice. Then, we centrifuged the lysed cells at 12 000 g for 10 minutes at 4°C to extract the supernatant for protein quantification. We utilized the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) to quantify protein concentration. The obtained supernatant was then boiled for 10 minutes by adding 5X SDS. Protein (50 μg) was added to the prepared 12% SDS‐PAGE gels for electrophoretic separation and transferred to 0.45 µm PVDF membranes (Amersham Hybond, GE Healthcare). We then utilized 1% albumin from bovine serum (Amresco) to block the PVDF membranes for 2 hours. Then, the membranes were incubated overnight with diluted xCT (1:1000, ab37185; Abcam) and β‐actin (1:1000, ab179467; Abcam) antibodies on a shaker at 4°C. We washed the membranes with TBS‐T (0.1% Tween‐20) at room temperature three times for 10 minutes. The goat anti‐rabbit IgG H&L (HRP) (1:2000, ab7090; Abcam) for 1 hour was utilized to incubate the membranes. After washed, the membranes were exposed to enhanced chemiluminescence substrate detection solution (Lulong Biotech) subsequently.

The protein concentration was measured using a bicinchoninic acid (BCA) assay (#23225; Pierce, USA) according to the manufacturer’s instructions. The primary antibodies used in this study are as followed: anti-KDM5B (1:500, Cell Signaling Technology, #15327), anti-H3K4me3 (1:1000, Cell Signaling Technology, #9751), anti-H3 (1:1000, Cell Signaling Technology, #4499), anti-CCNE1 (1:1000, Cell Signaling Technology, #20808), anti-CCNE2 (1:1000, ProteinTech, 11935-1-AP), anti-CDK2 (1:1000, ProteinTech, 10122-1-AP), anti-FLAG (1:1000, Cell Signaling Technology, #8146), anti-FBXW7 (1:1000, Abcam, ab109617), anti-FLI1 (1:1000, Abcam, ab133485), and β-actin (1:5000, Cell Signaling Technology, #4970). The secondary antibodies are goat anti‐rabbit immunoglobulin G (1:2000, Abcam, ab7090) and goat anti‐mouse immunoglobulin G (1:2000, Abcam, ab97040). The unprocessed western blot images were shown in Supplementary Fig. S2.

After CDDP treatment or transfection with miR-378a-3p mimics, NC mimics, pcDNA-IGF1R, and pcDNA, cells were digested and collected using 0.25% trypsin, washed twice using pre-chilled 1× PBS ( phosphate buffer saline), centrifuged, and supernatants were discarded. The total cellular protein was extracted and then separated by SDS-PAGE electrophoresis and transferred to the PVDF membrane. The cells were closed with 4% skim milk for 1 h, incubated overnight at 4°C with primary antibody dilution of IGF1R (ab182408, 1:1,500, Abcam, Cambridge, MA, USA), washed 3 times with 1× phosphate buffered saline Twen-20, and then incubated for 1 h using secondary antibody dilution (ab7090, 1:2,000, Abcam), and developed by adding ECL developer [14 (link)].

The retina of mice and BV-2 or HRECs were collected and homogenized with lysis buffer, followed which centrifugation (1600 × g, 15 min) was performed at 4°C. Protein concentration was quantified in supernatant fluid by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to the PVDF membranes (GE Healthcare Europe GmbH, Freiburg, Germany). Proteins were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies (p-p65: ab76302; t-p65: ab32536; occludin: ab216327; claudin-1: ab180158; ZO-1: ab216880; VEGF: ab214424; CD31: ab182981; GAPDH: ab8245. Abcam, England. p-IκB: #2859, t-IκB: #9242, Iba-1: #17,198, Cell Signaling Technology, USA). Subsequently, HRP-conjugated secondary antibody (ab7090, abcam, England) was used to incubate the membranes for 1 h at 37°C. Protein bands were visualized by enhanced chemiluminescence reagent (Millipore Corp., Bedford, MA).