Ultra high sensitivity ecl substrate kit

Western bolt analysis was applied to detect the protein expression levels of apoptosis indexes and exosomal markers. The BCA protein assay was adopted as a standard to determine the protein content. Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was blocked with 50% fat-free milk and incubated overnight with the appropriate primary antibodies including anti-Bcl-2 (sc-7382, 1:1000), anti-Bax (sc-7480, 1:1000), anti-β-actin (sc-8432, 1:5000) (SantaCruz Biotechnology, CA, USA), anti-cleaved caspase-3 (9668, 1:1000) and anti-cleaved caspase-9 (9509, 1:1000) (Cell Signaling Technology, Beverly, MA, USA). After fully rinsing with Tris Buffered Saline Tween containing 0.1% Triton×100 buffer solution, the membrane was incubated with secondary antibody (sc-69,786, 1:1000; Santa Cruz Inc, Santa Cruz) at room temperature for 1 h. Then, the protein bands were visualized by an Ultra High Sensitivity ECL Substrate Kit (ab133409, Abcam, Shanghai, China). The semi-quantitative results were obtained by quantification of optical density using the ImageJ software.

Lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% TritonX-100, 1% proteinase inhibitor cocktail, 1 mM phenylmethanesulfonyl fluoride (PMSF), pH 7.5) was used to extract total proteins. Apoplastic fluid was extracted as described [70 ]. Protein samples were boiled in 5 × sodium dodecyl sulfate (SDS) loading buffer for 10 min before loading on a gel for electrophoresis. The proteins were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane with transfer buffer. The membrane was then incubated with HRP-conjugated anti-Flag antibody (ABclonal, Wuhan, China) at RT for 3 h, and the blots were detected with Ultra High Sensitivity ECL Substrate Kit (Abcam, Cambridge, UK).

Proteins of xenografts and cells were extracted using Pierce™ IP lysis buffer (87787; Thermal Fisher). 40 μg of the protein was resolved in each well of 10% SDS-PAGE and transferred to Polyvinylidene Fluoride (PVDF) membranes. Then, the blots were blocked by 5% fat free milk and incubated with anti-HIF-1α (1:1000; ab1; Abcam), anti-PGC1α (1:1000; ab106814; Abcam), anti-MCT1 (1:3000; ab90582; Abcam), anti-MCT4 (1:1000; ab234728; Abcam), anti-CD174 (1:2000; ab235831; Abcam), anti-HMGB1 (1:1000; ab18256; Abcam), anti-E-cadherin (1: 20000; ab40772; Abcam), anti-N-cadherin (1:10000; ab76011; Abcam), anti-Bcl-2 (1:1000; ab32124; Abcam), anti-p-GSK (1:10000; ab75814; Abcam), anti-GSK (1:8000; ab32391; Abcam), anti-ERK (1:10000; ab184699; Abcam), anti-p-ERK (1:1000; ab201015; Abcam), anti-JNK (1:1000; ab179461; Abcam), anti-p-JNK (1:5000; ab124956; Abcam), anti-p38 (1:5000; ab170099; Abcam), anti-p-p38 (1:1000; ab178867; Abcam) primary antibodies at 4 °C overnight. The day after, rabbit anti mouse IgG secondary antibody (1:2000; ab6728; Abcam) was incubated with the membranes at room temperature for 2 h. GAPDH was used as control. Finally, protein bands were visualized using the Ultra High Sensitivity ECL Substrate Kit (ab133409; Abcam).