Nuclear and plasma protein isolation kit

Cells, exosomes, and tissues were lysed in RIPA buffer. Protein concentration was determined using the BCA assay kit (Pierce, USA). Sources and dilution factors of primary antibodies were: rabbit polyclonal CD63 (1:1000; Bioworld, USA), CD9 (1:1000; Bioworld), CD81 (1:1000; Epitomics, USA), PCNA (1:1000; Bioworld), BCL-XL (1:100; SAB, USA), BCL-2 (1:1000; Bioworld), Bax (1:1000; Bioworld), Cytochrome C (1:500; Abcam, USA), caspase-3 (1:500; Bioworld), IL-1β (1:500; Bioworld,), LC3B (1:500; Abcam), Beclin-1 (1:600; Proteintech, USA), mTOR (1:500; SAB), p-mTOR (1:500; SAB), 4EBP1 (1:200; SAB), p70S6K (1:200; SAB), and mouse monoclonal GAPDH (1:3000; Kang Chen, China). The nucleoprotein and plasma protein was separated by the nuclear and plasma protein isolation kit (Vazyme, China), with the primary antibody NF-kB-P65 in the nucleus (1:500; SAB), primary antibody nucleoprotein Histone (1:1000; SAB). After incubation with the primary antibodies overnight at 4 °C, membranes were washed three times with Tris-buffered saline with 0.05% Tween-20 and challenged with HRP-conjugated goat anti-rabbit or goat anti-mouse antibody (1:2000; Bioworld). Western blot was performed by Luminata™ crescendo western HRP substrate (Millipore, USA) and analyzed using MD Image Quant Software.

Cells were collected harvested, pulverized and lysed in RIPA buffer. The nucleoprotein and plasma protein was separated by the nuclear and plasma protein isolation kit (Vazyme, USA). Equal amounts of protein were loaded and separated on SDS-PAGE gel. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Sources and dilution factors of primary antibodies were: α-SMA (1:1000, abcam, USA), TGF-β (1:500, Bioworld, China), LC-3B (1:1000, abcam), Beclin-1 (1:1000, Proteintech), GAPDH (1:1000, Beyotime, China), NF-kB-P65 in the nucleus (1:500; SAB, China) and primary antibody nucleoprotein Histone H3 (1:1000; SAB). After incubation with the primary antibodies overnight at 4°C, membranes were incubated with HRP-conjugated goat anti-rabbit(1:1000, Beyotime, China), or goat anti-mouse antibodies (1:1000, Beyotime, China). This was followed by detection with luminata^TM crescendo western HRP substrate (Millipore, USA) and quantitated using MD Image Quant Software.