Mouse monoclonal anti vimentin

Western blot analysis was performed as previously described25 (link). The primary antibodies were as follows: mouse monoclonal anti-E-cadherin (1:2000, Proteintech, Rosemont, USA), mouse monoclonal anti-Vimentin (1:10000, Proteintech), rabbit polyclonal anti-MMP9 (1:500, Proteintech), rabbit polyclonal anti-VEGF (1:1000, Proteintech), rabbit polyclonal anti-ELF3 (1:500, Abcam, Cambridge, MA), rabbit polyclonal anti-Notch3 (1:500, Proteintech). Rabbit polyclonal anti-GAPDH (1:5000, Proteintech) was used as an endogenous control.

Western blot analysis was performed according to the description previously [20 (link)]. The following primary antibodies were used: rabbit polyclonal anti-ABCB1 (1: 500, Proteintech, Rosemont, USA), rabbit monoclonal anti-CCR7 (1:10,000, Abcam, Cambridge, MA), mouse monoclonal anti-ERK1/2 (1:2000, Proteintech), mouse monoclonal anti-P38 MAPK (1: 2000, Proteintech). rabbit monoclonal anti-phospho-p44/42 MAPK(Erk1/2)(Thr202/Tyr204, 1: 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-phospho-p38 MAPK (Thr180/Tyr182, 1: 1000, Cell Signaling Technology), mouse monoclonal anti-E-cadherin (1:2000, Proteintech), mouse monoclonal anti-Vimentin(1:5000, Proteintech), rabbit polyclonal anti-VEGF(1: 1000, Proteintech). Rabbit polyclonal anti-GAPDH (1:5000, Proteintech) was used as an internal control.

Female athymic four-week-old BALB/c nude mice were purchased from HFK Bio-Technology Co. Ltd. (Beijing, China). A total of 5 × 10⁶ MDA-MB-231 cells, resuspended in Opti-MEM, were injected into the mammary fat pad. Tumor volume was analyzed as V = D × d² × 0.5 (D, the longer diameter; d, the shorter diameter). When the tumor reached about 60 mm³, the mice were divided into a si-control group (n = 5) and a si-BCAR4 group (n = 5) randomly. The in vivo siRNA for BCAR4 knockdown was synthesized by RiboBio (Guangzhou, China). The sequence of the in vivo BCAR4 knockdown was as follows: si-BCAR4: CAC AAT TGA TGT TCT CTA A. In vivo si-BCAR4 or the control (2.5 nmol / 20 g body weight) was directly administered into the implanted tumor two times per week for three weeks. One month after the first injection of si-BCAR4 or the control, all mice were anesthetized with 1% pentobarbital sodium. The tumors, lungs, and livers were obtained and then fixed in formalin for hematoxylin and eosin (HE) staining or immunohistochemistry. The rabbit polyclonal anti-CCR7 (1:200, Proteintech) was used to determine CCR7 expression. The mouse monoclonal anti-E-cadherin (1:1000, Proteintech), mouse monoclonal anti-Vimentin (1: 1000, Proteintech), rabbit polyclonal anti-VEGF (1:200, Proteintech) was used to detect E-cadherin, Vimentin and VEGF expression respectively.