Chemiscope 3300 mini

Cell lysate or MB tissue homogenate was prepared in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail (Roche), and the total protein concentration was evaluated using a BCA Protein Assay Kit (Beyotime). Then, equal amounts of protein samples were loaded and separated by 10% SDS–PAGE (Beyotime) and transferred onto PVDF membranes (Millipore). The membranes were blocked with TBS containing 0.05% Tween-20 and 5% defatted milk powder and incubated overnight at 4 °C with primary antibodies against GFAP (1:200, BD Biosciences, 556330), GAPDH (1:5000, Proteintech, 60004), total p38 (1:1000, Cell Signaling Technology, CST, 9212), phosphorylated p38 (1:1000, CST, 9215), β-Tubulin (1:10,000, Proteintech, 66240), phosphorylated Erk1/2 (1:1000, CST, 4370) and total Erk (1:1000, CST, 4695). The following day, the membranes were incubated with the HRP (horseradish peroxidase)-conjugated secondary antibodies anti-mouse IgG (1:5000, CST, 7076) and anti-rabbit IgG (1:5000, CST, 7074) at room temperature for 2 h. Bands were visualized using high-signal ECL substrate (Beyotime) and exposed on a ChemiScope 3300 mini (Clinx).

The proteins were extracted using RIPA Lysis Solution (P0013 C, Beyotime, Shanghai, China) and protein concentrations were measured using a bicinchoninic acid protein assay kit (KeyGen Biotech Co., Ltd, Nanjing, China). The proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Roche, Switzerland). The membranes were incubated with the following primary antibodies: IFIT1 (ab236256, Abcam, USA), ATP6V0D2 (H00245972-M01A, Abnova, Shanghai, China), DC-STAMP (ab238151, Abcam), ATP6i (H00000525-M02, Abnova), CTSK (H00001513-M01, Abnova), TRAP (ab2721, Abcam), p-JAK1 (ab138005, Abcam), JAK1 (ab133666, Abcam), p-STAT3 (ab267373, Abcam), STAT3 (ab68153, Abcam), c-Fos (ab222699, Abcam), MMP9 (ab76003, Abcam), NFATc1 (GTX09510, GeneTex, USA), and β-actin (C1313, Applygen, Beijing, China). After a 12 h incubation, the membranes were incubated with the secondary antibody, and the intensity of protein expression was detected by ChemiScope 3300 Mini (CLINX, Shanghai, China) using enhanced chemiluminescence (Beyotime, Beijing, China).

The cells were collected and lysed with cell lysis buffer (Beyotime). Samples of the lysates were separated on 6%–15% SDS-PAGE gels and transferred to nitrocellulose filter membranes. The membranes were incubated with primary antibodies at 4 °C overnight, including Vimentin (1:1000, Cell signaling Technology, CST), E-cadherin (1:500, Santa Cruz), N-cadherin (1:500, CST), ZEB1 (1:500, CST), Snail (1:500, CST), PARP (1:1000, CST), pro-caspase 3 (1:1000, CST), GAPDH (1:2000, CST), followed by incubation with an HRP-conjugated anti-mouse or anti-rabbit secondary antibody separately. Finally, the bands were detected by ChemiScope 3300 Mini (Clinx) using the ECL substrate (Cyanagen).

Protein concentrations of EVs were evaluated using a BCA Protein Assay Kit (Beyotime, Haimen, China) according to the manufacturer’s instructions. Bovine serum albumin (BSA) was used as a standard. Antibodies against EV markers of CD9 (ab92726, 1:1000, Abcam, Cambridge UK), CD63 (ab134045, 1:1000, Abcam, Cambridge UK), and calnexin (ab22595, 1:1000, Abcam, Cambridge UK) were tested against anti-mouse secondary antibody (400108, 1:5000, BioLegend, San Diego, CA, USA) using Western blotting. Protein lysate (10 μg per well) was loaded onto 10% or 15% SDS-PAGE gels depending on the molecular weight of the target proteins. The separated proteins were transferred onto a methanol-activated polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). Membranes were blocked in a blocking buffer containing 5% skim milk powder in 1× TBST for 1 h at room temperature and then incubated with the primary antibodies for overnight at 4 °C. Excess primary antibodies were removed by washing with TBST three times. Next, the membranes were incubated with the secondary antibody for 1 h at room temperature, followed by incubation with an Immobilon™ Western Chemiluminescent HRP Substrate (ECL, Merck Millipore, Burlington, MA, USA) for 5 min at room temperature, for chemiluminescent signal detection using a ChemiScope 3300 mini (Clinx, Shanghai, China).