M1210 2

Cells were harvested and prepared using the Radio Immunoprecipitation Assay (RIPA) lysis buffer (Boster, China). Protein concentrations of the samples were determined using the bicinchoninic acid (BCA) protein assay (Millipore, Bedford, MA, USA). Each sample (30 μg) was boiled to denature the protein, and western blot analysis was performed. The primary antibodies used were polyclonal antibodies against NKTR (1:1,000, AAA515N, Invitrogen, Carlsbad, CA, USA) and β-actin (1:5,000, M1210-2, HuaBio, China).

The sequences of the primer pairs used in qPCR are as follows: Sag-Fwd: 5′-TG GAGGACGGCGAGGAAC-3′, Sag-Rev: 5′-CCCCAGACCACAACACAGTC-3′; Rbx1-Fwd: 5′-CTTTGTATCGAATGTCAGGC-3′, Rbx1-Rev: 5′-GTCACTAGACGAGTAACAG-3′; Ube2f-Fwd: 5′-GACTGTAAGCCCAGATGAG-3′, Ube2f-Rev: 5′-CCTTTAATGTTCTAGTGGG-3′; Ube2m-Fwd: 5’-CTGTCCTGATGAAGGCTTC-3′, Ube2m-Rev: 5′-GTTCGGCTCCAAGAAGAG-3′; Gapdh-Fwd: 5′-GCCGCCTGGAGAAACCTGCC-3′, Gapdh-Rev: 5′-GGTGGAAGAGTGGGAGTTGC-3′.
The antibodies used in western blot are as follows: anti-Ube2f, Proteintech, 17056-1-AP (dilution 1: 1000); and anti-β-Actin, HuaBio, M1210-2 (dilution 1: 5000).

RNA was isolated and reverse-transcribed to cDNA with oligos using the Takara reverse transcriptase system (RR047A), then analyzed using an SYBR Green master mix (E166, novoprotein). A table of human and mouse primers used in this study is shown in Table S1.
For western blotting, cells were lysed in 1× RIPA buffer (20-188, millipore) and processed by standard western blot techniques. Membranes were blocked with 5% BSA in phosphate buffer saline (PBS) containing 0.5% Tween-20 and incubated with primary antibodies for β-actin (1:1000, M1210-2, Huabio), caspase-8 (1:1000, 66093-1-Ig, Proteintech), caspase-9 (1:1000, 10380-1-AP, Proteintech), IL-23A (1:200, bs-1193R, Bioss).

Cells were lysed with the mixture containing cell lysis buffer for Western and IP and 1% phenylmethanesulfonyl fluoride (PMSF) (Biosharp, Beijing, China) on ice to extract protein. Protein samples were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. And the membranes were blocked with 5% non-fat milk at room temperature for 1 h, then incubated with the primary antibody overnight at 4 °C and next with the secondary antibody for 1 h at room temperature. The protein bands were visualized using ECL Protect from Light (Biosharp) and quantified using Image J software. The primary antibodies used in this study were as follows: ADIPOQ (sc-136131, Santa Cruz, Watsonville, CA, USA, diluted 1:200), FLAG (20543–1-AP, Proteintech, Rosemont, IL, USA, diluted 1:1,000), YTHDF1 (17479–1-AP, Proteintech, diluted 1:1,000), GFP (ET160–25, Huabio, Hangzhou, China, diluted 1:5,000), β-actin (M1210–2, Huabio, diluted 1:5,000). The secondary antibodies were as follows: goat anti-mouse IgG-HRP (HA1006, Huabio, diluted 1:2,000), goat anti-rabbit IgG-HRP (HA1001, Huabio, diluted 1:2,000).

Total RNA of LPS activated and resting BV-2 cells were isolated using TrizolTM reagent (Life Technologies, CA, USA). Reverse transcription and qRT-PCR were conducted separately using a reverse transcription kit (Takara) and SYBR Green qPCR SuperMix kit (Invitrogen, Thermo-Fisher) according to the manufacturer’s protocol. The list of all primer pairs used for qRT-PCR is shown in Table 1 in Supplementary Material.
Western blot with transfected cells and retinas were conducted as described previously^{48 (link)}. After centrifugation of cell lysates, protein was quantified with the BCA assay and then loaded onto an 8–13% SDS-PAGE gel. Primary antibodies used in this study were as follows: rabbit anti-PI3K p85 alpha (1:1000, ET1608-70, HUABIO), mouse anti-Pik3ip1 (1:1000, sc365777, Proteintech), rabbit anti-Akt (1:1000, 4685, Cell Signaling Technology)), rabbit anti-pAkt (1:1000, AF3262, Affinity), mouse anti-Beta tubulin (1:1000, EM1701-59, HUABIO), rabbit anti-VEGFA (1:1000, 50661S, Cell Signaling Technology), mouse anti-FGF2 (1:1000, SC-365106, Santa Cruz Biotechnology), mouse anti-HGFα (1:1000, SC-374422, Santa Cruz Biotechnology), mouse anti-MMP9 (1:1000, SC-13520, Santa Cruz Biotechnology), mouse anti-PDGFβ (1:1000, SC-365805, Santa Cruz Biotechnology), mouse anti-TGFβ1 (1:1000, SC-130348, Santa Cruz Biotechnology) and mouse anti-βactin (1:1000, M1210-2, HUABIO).

Cells were treated with various compounds, and the protein levels were measured by standard Western blotting analysis. The antibodies used were obtained from the following vendors: CUL-5 (sc-373822, Santa Cruz/SC), CUL-1 (sc-11384, SC), CUL-2 (ab166917, Abcam), CUL-3 (2759S, Cell Signaling Technology/CST), CUL-4A (2699S, CST), CUL-4B (12916-1-AP, Proteintech), NOXA (OP180, EMD Millipore), UBE2F (sc-398668, SC), UBE2M (sc-390064, SC), PARP (9542S, CST), Caspase 3 (9662S, CST), CUL-5 CTD (AV35127, Sigma-Aldrich), CUL-1 CTD (12895-1-AP, Proteintech), NEDD8 (ab81264, Abcam), p-ATR T1989 (2853S, CST), ATR (2790S CST) p-RPA32 S33 (ab211877, Abcam), RPA32 (ab2175, Abcam), p-CHK1 S345 (2348, CST), CHK1 (sc-8408, SC), p-ATM S1981 (5883S, CST), ATM (2873S, CST), p-CHK2 T68 (2197P, CST), CHK2 (6334S, CST), γH2AX (05-636, EMD Millipore), DAPI (D1306, Invitrogen), β-Actin (M1210-2, HuaBio) and α-Tubulin (T8203, Sigma-Aldrich).