Fig 2: Gab1 and Grb2 are involved in pY772-EphA2-activating Shp2/Erk-1/2 signaling pathway in the NPC cells.a Western blotting showing the levels of p-Shp2 (Y542/Y580) and p-Erk-1/2 in the EphA2-WT-expressed NPC cells infected with the lentiviral vector expressing Gab1 shRNA, and their control cells. b Co-IP showing the levels of p-Shp2(Y542/580) and Shp2 bound to EphA2 in the EphA2-WT-expressed NPC cellsinfected with the lentiviral vector expressing Gab1 shRNA, and their control cells. Total cell proteins from the infected cells were prepared and subjected to immunoprecipitation immunoprecipitation (IP) with anti-EphA2 antibody followed by immunoblotting with antibodies against p-Shp2 (Y542/Y580), Shp2, or EphA2. c Co-IP analysis showing the levels of Grb2 bound to Shp2 in the EphA2-WT-expressed NPC cells infected with the lentiviral vector expressing Gab1 shRNA, and their control cells. Total cell proteins from the infected cells were prepared and subjected to immunoprecipitation (IP) with anti-Shp2 antibody followed by immunoblotting with antibodies against Grb2 or Shp2. d Co-IP showing the levels of p-Gab1, Gab1, and Grb2 bound to Shp2 in the NPC cells expressing EphA2-WT or EphA2-YA and their control cells. Total cell proteins from the indicated cells were prepared and subjected to immunoprecipitation with anti-Shp2 antibody followed by immunoblotting with antibodies against p-Gab1, Gab1, Grb2, or Shp2. e A model for pY772-EphA2-dependent NPC cell growth. EphA2 is phosphorylated at Y772 by growth factors in a ligand-independent manner and phosphorylation of EphA2 at Y772 (pY772-EphA2) activates Shp2/Erk-1/2 signaling, and both Gab1 and Grb2 are involved in pY772-EphA2-activating Shp2/Erk-1/2 activation, which promotes NPC cell growth. EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibits pY772-EphA2, which blocks NPC cell growth.

Fig 3: Protein expression levels of Gab1, TPO, and c-Mpl in rats from each group (first lane, control; second lane, model; third lane, low-dose; fourth lane, medium-dose; fifth lane, high-dose). ?Comparison with blank group, P < 0.05. *Comparison with the model group, P < 0.05.

Fig 4: Expression of GAB1 in ASO patients. (A) Sclerotic intima was collected from four patients clinically diagnosed with ASO and the normal intima obtained from amputation patients was used as a control. The tissues were homogenized in RIPA buffer with a complete protease inhibitor. The lysates were processed for Western blotting for detecting the protein expression level of GAB1. The protein expression level of GAPDH was used as a loading control. (B) Protein levels of GAB1 were quantitated by means of densitometric analysis using Image J normalized to GAPDH and presented as fold changes compared with the control group (mean ± standard deviation, n = 4). *P < 0.05. (C) Immunocytochemical staining was conducted using anti-GAB1 (Red). Nuclei were counterstained by DAPI (Blue). Scale bars: 10 µm. ASO: Arteriosclerosis obliterans; DAPI: 4',6-Diamidino-2-phenylindole; GAB1: GRB2 associated binding protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RIPAA: Radioimmunoprecipitation assay.

Fig 5: GAB1 natively regulates autophagy. (A) HUVECs were treated with either control siRNA (siControl) or GAB1-targeting siRNA (siGAB1) for 48 h, followed by treatment of HBSS for 6 h for the induction of autophagy presented with or without BAF. Cell lysates were harvested to examine the protein levels of GAB1 (using anti-GAB1 antibody), LC3I/II, and ß-actin by Western blotting. (B) HeLa cells were transiently transfected with control or GAB1 expression vector control for 24 h, followed by the treatment as mentioned above. Western blotting was performed and analyzed as mentioned above. (C) Protein levels of LC3II were quantitated by densitometric analysis using Image J normalized to ß-actin and presented as fold changes compared with the control group. *siControl vs. siGAB1 in HBSS- and BAF-; †siControl vs. siGAB1 in HBSS+ and BAF-; ‡siControl vs. siGAB1 in HBSS+ and BAF+. (D) Protein levels of LC3II were quantitated by densitometric analysis using Image J normalized to ß-actin and presented as fold changes compared with the control group. *Control vs. GAB1 in HBSS- and BAF-; †Control vs. GAB1 in HBSS+ and BAF-; ‡Control vs. GAB1 in HBSS+ and BAF+. ASO: Arteriosclerosis obliterans; BAF: Bafilomycin A1; DAPI: 4',6-Diamidino-2-phenylindole; GAB1: GRB2 associated binding protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HBSS: Hank balanced salt solution; HUVEC: Human umbilical vein endothelial cells.