Bca protein assay kit

Cells were lysed using M-PER Mammalian Protein Extraction Reagent lysis buffer (Thermo Fisher Scientific, United States) and protease inhibitor cocktail (Bimake, China). The BCA protein assay Kit (Epizyme, China) was used to quantitate total protein levels. Proteins were separated by sodium dodecyl sulfate−10% polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinyl difluoride (PVDF) membranes (Millipore, United States). The membranes were blocked for 2 h at room temperature in TBST with 5% non-fat milk and incubated overnight at 4°C with primary antibodies including anti-β-actin (AF7018, 1:3,000, Affinity), anti-DLL3 (ab103102, 1:1,000, Abcam), anti-METTL3 (ab195352, 1:1,000, Abcam), anti-NOTCH3 (5278T, 1:1,000, Cell Signaling Technology), and anti-HES1 (11988S, 1:1,000, Cell Signaling Technology). The membranes were washed three times with TBST and incubated with anti-rabbit IgG HRP-linked antibody (31466, 1:3,000, Thermo Fisher Scientific) in room temperature. Finally, the ChemiDoc XRS + system (Bio-Rad, United States) was used to detect protein signal by Immobilon Western Chemiluminescent HRP substrate (Millipore, United States). The quantifications were evaluated using ImageJ software.

Pancreatic tissue, cells, or EV pellets were lysed in radioimmunoprecipitation assay lysis buffer (RIPA, PC101, Epizyme, China) containing 1% Protease Inhibitor Mix (GRF101, Epizyme, China) and the protein concentration obtained after centrifugation was measured using the BCA Protein Assay Kit (ZJ101, Epizyme, China). The same amount of each protein sample (30 µg/lane) was separated by electrophoresis and transferred to nitrocellulose filter membranes. The membranes were then blocked with 5% milk in TBST for 1 h at room temperature and then incubated with primary antibody (Table S2, Supporting Information) overnight at 4 °C. Then, the membranes were incubated with the secondary antibody for 1 h at room temperature. Immunoreactive protein bands were visualized with an enhanced chemiluminescence system (Amersham Imager 600; GE Healthcare Bio‐Sciences Corp., USA) or an Odyssey M Infrared Imaging System (LI‐COR Biosciences, USA) and the bands were analyzed for grey scale values using ImageJ software (National Institutes of Health, USA).

Protein was extracted by RIPA lysis buffer with Protease and Phosphatase Inhibitor Cocktail, and protein concentration were measured by the BCA Protein Assay Kit (ZJ102, Epizyme). Proteins of each sample was separated by sodium dodecyl sulfate polyacrylamide gel and transferred onto PVDF membranes. After blocking by 5% skim milk in TBS-T at room temperature, the membranes were incubated with primary antibody at 4 °C overnight, including LRRK2 (91882, Cell Signaling Technology) and GAPDH (as a reference, HRP-60004, Proteintech). The membranes were incubated with HRP-linked secondary antibody and detected by chemiluminescence.

Tissues, cells, or EVs were lysed and extracted by RIPA lysis buffer (Epizyme, China) for 15 min on ice. After centrifugation at 15,000 × g for 15 min at 4 °C, the concentration of protein supernatant was determined using the BCA protein assay kit (Epizyme, China).

RIPA lysis buffer (Epizyme) was used to lyse cells. After centrifugation, the supernatant was collected in a clean tube. BCA Protein Assay Kit (Epizyme) was applied to quantify protein concentrations. Subsequently, loading buffer (Epizyme) was mixed with supernatant and heated. Samples of equal amounts were separated by 10% PAGE Gel Fast Preparation Kit (Epizyme) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). PVDF membranes were blocked with skimmed milk powder (Servicebio) dissolved in TBST at room temperature for 2 h. The membranes were incubated with primary antibodies against VISTA (1:1000, CST) and GAPDH (1:10 000, CST; Table S2) at 4°C overnight. GAPDH was used as control. After incubation with secondary antibodies (1:5000, Servicebio), the signals were visualised by the ECL system (Clinx Science Instruments). Experiments were performed in triplicate.

Total protein was extracted and quantified using a total protein extraction kit (KeyGen Biotech, Nanjing, China) and BCA protein assay kit (EpiZyme, Shanghai, China), respectively, according to the manufacturer’s instructions. In total, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked at room temperature for 30 min using protein-free rapid blocking buffer (EpiZyme), followed by the addition of primary antibody and incubation overnight at 4°C with gentle shaking. The primary antibodies included anti-actin beta (ACTB, 1 : 3000; 205 361-AP, Proteintech), anti-microtubule associated protein 1 light chain 3 beta (MAP1LC3B/LC3B, 1 : 1000; ab48394, Abcam, Cambridge, UK) and anti-p62/sequestosome 1 (SQSTM1, 1 : 10,000; ab56416, Abcam). The membranes were washed three times with TBST for 5 min and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 10,000; SA00001–2, Proteintech) for 2 h at 25°C. Finally, color development and quantification were performed.

Western blot analyses of the kidney and intestine tissues were conducted following a previous protocol [34 (link)]. Kidney and intestine tissue proteins were extracted and protein concentrations were determined by the BCA protein assay kit (Epizyme, Shanghai, China). Thirty μg of total proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were closed with 5% skim milk powder for 2 h, incubated with primary antibody at 4 °C overnight, and then incubated with secondary antibody. Bands were visualized using WesternBright ECL (ABclonal, Wuhan, China) with ChemiDoc XRS + Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The grayscale values of the bands were calculated and normalized to the grayscale values of β-actin.