Ripa lysis and extraction buffer

To prepanre cell lysates, RIPA Lysis and Extraction Buffer (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) which contained 10% protease inhibitor (Thermo Scientific, USA) were used to extract cell lysates by incubated on ice for 30 min, and then centrifuged at 14000 x g for 15 min at 4 °C. The protein concentrations were determined by BCA kit (Thermo Scientific, USA). For western blotting, we used the protocol which was performed previously [31 (link)]. In brief, we first mixed supernatants with 4x SDS-PAGE sample loading buffer, and they were denatured at 95 °C for 10 min. Second, the proteins were separated by SDS-PAGE gel, transferred to a polyvinylidene difluoride membranes, incubated with specific primary antibodies at 4 °C overnights, and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using a VersaDoc Image System (BioRad, Hercules, CA, USA). For immunoprecipitation, the lysate was treated using the Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) according to the protocol. The final precipitated proteins were analyzed via western blotting with the corresponding antibodies.

Mammary gland tissues were solubilized in RIPA Lysis and Extraction Buffer (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to extract total protein. After boiling for 5–10 min, the protein samples extracted from different cell suspensions were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk prepared in Tris-buffer and incubated with the primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. The membrane was then incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), for 4 h at room temperature. The blot was developed using the ECL™ Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ, USA), and the proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA).

Cultured cells were collected and lysed in ice-cooled RIPA Lysis and Extraction Buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Bicinchoninic acid assay (Nanjing KeyGen Biotech Co., Ltd; Nanjing, China) was employed to determine the total protein concentration. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% gel) was performed for the separation of proteins with equivalent concentrations, following which they were transferred to polyvinylidene fluoride membranes. Blocking was performed by incubating the imprinted membranes for 2 h at room temperature with 5% nonfat dried milk diluted in Tris-buffered saline containing 0.1% of Tween 20. After overnight incubation with primary antibodies at 4°C, a horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; Abcam, Cambridge, UK) was diluted at a concentration of 1:5000 and further used for incubating the membranes at room temperature for 1 h. The protein bands were developed using ECL Western Blotting Substrate kit (Abcam). The primary antibodies and their sources are as follows: ITGB1 (cat. no. ab52971; Abcam) and GAPDH (cat. no. ab181602; Abcam). GAPDH was used as the loading control.

Total proteins from exosomes or cells were isolated using the RIPA Lysis and Extraction Buffer (Invitrogen) according to the manufacturer's protocol. The proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which they were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Massachusetts, USA), and then blocked with 5% skim milk. The PVDF membranes were incubated with VCAM-1 (Cell Signaling Technology, CST, Boston, USA, #32653); ICAM-1 (CST, #4915); CD63 (Abcam, Shanghai, China, ab134045); KLF2 (Abcam, ab203591); or GAPDH (CST, #5174) antibodies overnight at 4°C, and then incubated with secondary antibodies (goat anti-rabbit or mouse) (ZSGB-BIO, Beijing, China) for 1 h. The PVDF membranes were then exposed in a chemiluminescence instrument (Bio-Rad ChemiDoc XRS+, USA) using the SuperSignal West Dura Extended Duration Substrate Kit (Thermo, Scientific, Beijing, China).

Interested cells were lysed in cold RIPA Lysis and Extraction Buffer (Invitrogen, Gaithersburg, MD, USA) containing a protease inhibitor (Selleck, Houston, TX, USA). After centrifugation, equal amounts of the protein were loaded for electrophoresis on 10% denaturing SDS-PAGE gels. The blots were probed with the primary antibodies overnight at 4 °C, followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. CD68 (1:1000, CST, Beverly, MA, USA), CD206 (1:1000, CST, Beverly, MA, USA), E-Ca (1:1000, CST, Beverly, MA, USA), N-Ca (1:1000, CST, Beverly, MA, USA) (1:1000, CST, Beverly, MA, USA), snail (1:1000, CST, Beverly, MA, USA), slug (1:1000, CST, Beverly, MA, USA), p27kip (1:1000, CST, Beverly, MA, USA), pAKT (1:1000, CST, Beverly, MA, USA), pSTAT3 (Tyr705, 1:1000, CST, Beverly, MA, USA) or GAPDH (1:1000, CST, Beverly, MA, USA) were used in this study.