Gel image system

Protein (60 μg) was separated using 10% SDS‐PAGE, which was then transferred onto PVDF membranes. Then, the membranes were blocked with 5% (w/v) non‐fat milk. After blocking, the membranes were incubated with monoclonal primary antibody against vimentin (sc‐51721, 1:800; Santa Cruz, CA, USA), E‐cadherin (ab15148, 1:800; Abcam), Slug (ab27568, 1:600; Abcam), SOX4 (1:800; Abcam), N‐cadherin (sc‐59987, 1:1000; Santa Cruz) and anti‐GAPDH (ab14247, 1:2000; Abcam) at 4°C overnight. After incubating with secondary antibodies (Sigma) at room temperature for 60 minutes, protein bands were detected with the gel image system (Bio‐Rad).

Total protein concentration in liver was determined with a BCA Protein Quantification Kit (E112-02, vazyme, Nanjing, China). After boiling denaturation, total protein (35 μg) was separated by 10%~12% SDS-PAGE, then transferred to PVDF membranes (ISEQ00010, Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated (overnight at 4°C) with the following primary antibodies: Rabbit anti-indoleamine 2,3-dioxygenase 1 (IDO-1, 1:1000, 13268-1-AP, Proteintech, Wuhan, China), Rabbit anti-TDO-2 (1:500, 15880-1-AP, Proteintech, Wuhan, China), Rabbit anti-AADAT (KAT2, 1:500, A13090, ABclonal, Wuhan, China) and Rabbit anti-GAPDH (1:8000, ET1601-4, Huabio, Hangzhou, China). Then the HRP conjugated goat anti-rabbit IgG was incubated for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) reagent (P90720, Millipore, USA) and Gel Image System (Bio-Rad, Hercules, CA, USA). Target signals were analyzed by using Image J software (NIH, Bethesda, MA, USA).

The samples were homogenized by RIPA lysis buffer that contains proteinase inhibitors (Sigma) to extract total proteins. The proteins were quantified by BCA assay kit (Beyotime, China). An equal amount of proteins (30 μg) were divided on SDS-PAGE gel, shifted to NC membranes, blocked by 5% silk milk and hatched with specific primary antibodies against HMGB2 (1: 500), Bax (1: 500), Bcl-2 (1: 500), Caspese-3 (1: 500), and β-Actin (1: 1000) at 4°C overnight. Next day, the protein bands were incubated with specific HRP-conjugated anti-mouse and anti-rabbit secondary antibodies at room temperature for 45 minutes, visualized by using an ECL solution (Millipore, Germany) in a Gel Image system (Bio-Rad, Hercules, CA, USA). HMGB2, Bax, Bcl-2, Caspese-3 and β-Actin antibody were all acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

RIPA buffer (CST, USA) was used to extract the total protein, followed by the quantification based on the BCA method (Abbkine, USA). The proteins at same concentration were subjected in SDS-PAGE and transferred (PVDF, Millipore, USA), followed by the incubation with 5% milk and with the primary antibodies at 4°C overnight. The corresponding second antibodies (Boster, China) were used for incubating the membranes 1 hour at room temperature, followed by the visualization by using chemiluminescence detection kit (Beyyotime, China). The primary antibodies applied in this study comprising Bax (1:2000, ab32502, Abcam, USA), Bcl-2 (1:2000, ab32124, Abcam, USA), caspase-3 (1:2000, ab32351, Abcam, USA), and β-actin (1:2000, ab 8226, Abcam, USA). The grayscale of the images was quantified and calculated using a gel image system (Bio-Rad, USA) and the relative level of each band was normalized to the level of β-actin.

Mice were sacrificed to collect the brain and frozen at -80°C. Brain tissues were lysed, and centrifuged at 12000 rpm and 4°C for 20 min to obtain the total protein in the supernatant. The protein concentrations were estimated by the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Rockford, IL, USA). Then, the protein was boiled in sodium dodecyl sulfate (SDS)-loading buffer at 95°C for 10 min to denature. The appropriate amount of protein was run on a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm polyvinylidene fluoride membranes, and the membranes were blocked with 5% fat-free milk at room temperature for 1 h. The membranes were incubated at 4°C overnight with primary antibodies and incubated at room temperature for 1 h with the HRP-conjugated secondary antibody. Finally, an enhanced chemiluminescence reagent (Thermo Scientific Rockford) was used to develop the blots with a gel image system (Bio-Rad, CA, USA), and the analysis is performed by using ImageJ 1.49v (National Institutes of Health, USA, https://imagej.en.softonic.com/). The following antibodies were used: anti-TH antibody (Cat# ab112, Abcam, Cambridge, MA, USA), anti-β-actin antibody (Cat# 8457S, Cell Signaling Technology, Berkeley, CA, USA), anti-α-syn antibody (Cat# ab212184, Abcam), goat anti-rabbit antibody (Cat# ab6721, Abcam),.

Total protein was extracted by RIPA (Beyotime, Shanghai, China) with PMSF (Solarbio, Beijing, China). Then, the supernatant was collected and measured with a BCA protein assay kit (Cwbiotech, Beijing, China). Protein was separated by SDS-polyacrylamide gel electrophoresis (Thermo Scientific, Waltham, MA, USA) and blotted onto polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). Then, the PVDF membranes were blocked in 5% skim milk in TBS-T buffer at room temperature for 1 h, followed by incubation with primary antibody at 4 °C overnight. Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. The antibodies used in the trial are listed in Supplementary Table S5. Protein bands were visualized by a Gel Image System (Bio-Rad, Hercules, CA, USA).