Anti human sema3f

Nuclear and cytoplasmic fractions were isolated using NE-PER ^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA). Western blotting was carried out by standard procedures as previously described. ^{[13 (link)]} The primary antibodies used for the assay were as follows: anti-human SEMA3F (1:1000) (Chemicon, Temecula, CA, USA), anti-human MST2, MST1, MOB1, p-MOB 1, LATS1/2, YAP/TAZ, cleaved caspase-3, BCL2, p-YAP Ser397, p-YAP Ser127, and CCND2 (1:500) (Cell Signaling Technology, Danvers, MA, USA), anti-human β-actin (1:1000) (Cell Signaling Technology), anti-human CD20 (1:1000) (Abcam Cambridge, MA, USA), anti-human TAZ (1:1000) (Abcam), and anti-human Lamin B1 (1:1000) (Novus Biologicals, Littleton, CO, USA).

Cells on glass coverslips were fixed for 20 min with 4% paraformaldehyde without permeabilization. After being washed three times with phosphate buffer saline (PBS) for 5 min each time, the fixed cells were then incubated in a protein-blocking solution for 20 min at room temperature. The primary antibody against SEMA3F (Chemicon) and CD20 (Abcam) was added, and the mixtures were incubated overnight at 4℃, followed by incubation with Alexa Fluor 647 or 488 (Invitrogen) at 37℃ for 30 min. The cells were counterstained with Hoechst 33342 to reveal the nuclei. All samples were then analyzed by Confocal laser scanning microscope (CLSM LSM 800, Zeiss, Oberkochen, Germany).
IHC staining was performed using the Dako REAL Envision Detection system (DAKO, CA, USA) according to the manufacturer's protocol. The primary antibodies used for IHC were anti-human TAZ (1:400, Abcam) and anti-human SEMA3F (1:500, Chemicon). Semiquantification of TAZ and SEMA3F expression was performed independently by two histopathologists according to the staining intensity and percentage of positive tumor cells in a blinded manner.