Anti lrg1

The protein concentration of EVs was measured by a Qubit Kit (Thermo Fisher Scientific Inc., MA, USA) and the protein mixture was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, the proteins were separated using a precast polyacrylamide mini-gels (Tri-glycine pH 8.3) with a Mini Trans-Blot module (Bio-Rad Laboratories Inc., CA, USA). Then, the proteins on gels were then electrically transferred onto polyvinylidene fluoride membrane, which were then blocked in PBST containing 5% fat-free milk powder. After that, the membranes were incubated with the primary antibody overnight at 4 ^oC. The following antibodies were diluted by blocking liquid for western blot analysis, including anti-CD63, anti-CD9, anti-LRG1 (Abcam plc, Cambridge, UK), and anti-CD81 (Santa Cruz Biotechnology Inc., CA, USA). Thereafter, the membrane has been washed incubated with the secondary antibody (HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG). Finally, the secondary antibody was washed by PBST 3 times, we used the enhanced chemiluminescence for immunodetection (PeiQing Science & Technology Co. Ltd., Shanghai, China) for imaging.

Cell pellets were lysed with RIPA buffer containing protease/phosphatase inhibitor cocktail (catalog number: 4693116001; Roche, Germany) and protein concentration was measured by BCA kit. Equal amounts of total protein were subjected to SDS-PAGE, followed by blocking with 5 % fat-free milk. Primary antibodies were incubated at 4 °C overnight, followed by secondary antibodies for 40 min at room temperature. All blots were developed using a chemiluminescence kit (SuperSignal ECL Kit, Thermo Fisher, USA). The following antibodies were used: anti-LRG1 (catalog number: #178698, 1:1000 dilution) was purchased from Abcam (Cambridge, MA). Anti-Tubulin (catalog number: #T4026S, 1:1000 dilution) was purchased from Sigma. Anti-phospho-STAT3(y705) (catalog number: #9541 S, 1:1000 dilution), anti-STAT3 (catalog number: #9139 S, 1:1000 dilution), anti-N-cadherin (catalog number: #13116t, 1:1000 dilution), anti-E-cadherin (catalog number: #3195 T, 1:1000 dilution), and anti-Slug (catalog number: #C19G7, 1:1000 dilution), Anti-phospho-Smad1/5(Ser463/465) (catalog number: #9516, 1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA). Anti-Twist1 (catalog number: #abs131127-50μg, 1:1000 dilution) was purchased from Absin (China).

Tissues lysates were electrophoresed on SDS-PAGE gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk and the primary antibodies were used for incubation overnight at 4 °C. After washing, PVDF membranes were incubated with secondary antibodies for 1 h. Immunoreactive bands were quantitatively analyzed with ImageJ software (http://imagej.nih.gov/ij/) [24 (link)]. The primary antibodies were as follows: anti-LRG-1, 1:1000; anti-Smad1/5, 1:1000 (Abcam, Cambridge, UK); anti-GAPDH, 1:10000; anti-MMP-2, 1:1000; anti-MMP-9, 1:1000; anti-TIMP-1, 1:1000; anti-JNK, 1:1000; anti-p-JNK, 1:1000; anti-ERK, 1:1000; anti-p-ERK, 1:1000; anti-p38, 1:1000; anti-p-p38, 1:1000; anti-p-Smad1/5, 1:1000 (Cell Signaling Technology, Beverly, MA, USA).