Hsp70

Total protein was extracted with RIPA buffer and boiled for degeneration. 30 μg of the protein were loaded into 7.5% SDS-PAGE. After being electrotransferred and blocking with 3% BSA, the membranes were incubated with primary antibodies. Rabbit against MUC1, CD63, E-cadherin, N-cadherin (1:1000; Proteintech, USA), hTERT (1:1000; Bioss, China), HSP70 (1:500; Boster, USA), TSG101 (1:1000; ABclonal, USA), β-actin (1:3000; Proteintech, USA) and mouse against CD9 (1:1000; Proteintech, USA) were used as primary antibodies. Anti-rabbit or anti-mouse IgG conjugated with HRP (1:5000; Abbkine, USA) was used as the secondary antibody. Enhanced chemiluminescence (Abbkine, USA) was used to display the bands.

Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer. The cell lysate was then quantified and boiled for 5 min before 30 μg of the samples were separated by10% SDS-PAGE. Proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked for 2 h with 3% BSA at room temperature. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies for the detection of NF-κB2 (1:1,000; Proteintech, USA), MMP2 (1:1,000; Proteintech), HSP70 (1:500; Boster, USA), CD9 (1:1,000; Proteintech), FAP-1 (1:1,000; Abbkine, USA), vimentin (1:1,000; Proteintech), α-SMA (1:1,000; Proteintech), and β-actin (1:3,000; Proteintech). The membranes were then washed (3 × 10 min) with Tris-buffered saline containing Tween-20 (TBST). After incubation for 90 min at room temperature with anti-rabbit secondary detection antibodies (1:5,000; Proteintech), immunoreactive bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Total proteins from M. amblycephala liver, spleen, brain, gill, and kidney (n = 5 for each group) were quantified and separated on 8% SDS-PAGE, then transferred to nitrocellulose membranes. The membranes were incubated for 2 h with polyclonal antibodies, Hif-1α (1:500) and Hsp70 (1:200) (Boster, China), respectively, and then anti-rabbit IRDye 800CW-labeled secondary antibody (1:10,000) at room temperature for 1 h, and observed using Odyssey Fc machine (Licor Biosciences, USA). Gray values of every band were further calibrated and measured by ImageJ 1.46r (NIH, USA).