Fig 1: Silencing of PTK6 decreases GAB1 expression. (A) RT-qPCR and (B) western blotting, respectively, determined GAB1 expression in immortalized human normal cervical epithelial cells (Ect1/E6E7) and cervical cancer cell lines (SiHa, HeLa, Ca-Ski, C-33A). (C and D) Co-IP assay was performed using control IgG beads and immunoblotted for PTK6 and GAB1. The expression of GAB1 (E) mRNA and (F) protein levels following PTK6 interference in cervical cancer C-33A cells. ***P<0.001 vs. Ect1/E6E7 or siRNA-NC. PTK6, protein tyrosine kinase 6; GAB1, GRB2-associated binding 1; RT-qPCR, reverse transcription-quantitative PCR; Co-IP, co-immunoprecipitation; siRNA, small interfering RNA; NC, negative control.
Fig 2: Circ_0061012 up-regulates the level of GAB1 through sponging miR-194-5p in IL-22-induced HaCaT cells(A,B) The mRNA and protein levels of GAB1 were measured in HaCaT cells treated with IL-22, IL-22 + si-NC, IL-22 + si-circ_0061012, IL-22 + si-circ_0061012 + anti-miR-NC or IL-22 + si-circ_0061012 + anti-miR-194-5p by qRT-PCR and Western blot assay. (C) The diagram revealed that circ_0061012 accelerated psoriasis progression through miR-194-5p/GAB1 axis. The experiments were independently repeated for three times with at least three technical repetitions. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 3: MiR-194-5p suppresses IL-22-induced progression of psoriasis partly through targeting GAB1 in HaCaT cells(A–E) HaCaT cells transfected with miR-NC, miR-194-5p, miR-194-5p + pcDNA or miR-194-5p + GAB1 were treated with 100 ng/ml IL-22 for 24 h. (A) Western blot assay was utilized to measure the protein level of GAB1 in HaCaT cells. (B) MTT assay was used to evaluate cell viability of HaCaT cells at specific time points, and cell proliferation curve was plotted to evaluate cell proliferation capacity. (C,D) Transwell migration and invasion assays were used to measure the abilities of migration and invasion of HaCaT cells. The numbers of migrated and invaded HaCaT cells (stained by Crystal Violet) in five random fields were counted. Scale bar = 100 µm. (E) Western blot assay was used to measure the levels of Ki67 and MMP9 in HaCaT cells. The experiments were independently repeated for three times with at least three technical repetitions. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 4: Overexpression of GAB1 reverses the inhibitory effect of PTK6 interference on the invasion and migration abilities of cervical cancer cells. (A) Representative images of wound healing assay (magnification, ×100) and quantification of cell migration. (B) Representative images of Transwell assay (magnification, ×100) and quantification of cell invasion. (C) RT-qPCR and (D) western blotting were used to assess MMP12 and MMP9 expression levels. *P<0.05 and ***P<0.001 vs. Control or siRNA-PTK6 + Ov-NC. GAB1, GRB2-associated binding 1; PTK6, protein tyrosine kinase 6; RT-qPCR, reverse transcription-quantitative PCR; MMP, matrix metalloproteinase; siRNA, small interfering RNA; Ov, overexpression; NC, negative control.
Fig 5: Overexpression of GAB1 reverses the inhibitory effect of PTK6 interference on the proliferation of cervical cancer cells. (A) RT-qPCR and (B) western blotting respectively, detected GAB1 expression in cervical cancer C-33A cells. ***P<0.001 vs. Ov-NC. (C) C-33A cell proliferation was detected by CCK-8 assays. ***P<0.001 vs. Control; ##P<0.01 and ###P<0.001 vs. siRNA-PTK6 + Ov-NC. (D) TUNEL staining assays detected the apoptosis of C-33A cells. (E) Western blotting was used to assess the levels of Bcl-2, Bax and Birc5 in C-33A cells. **P<0.01 and ***P<0.001 vs. Control or siRNA-PTK6 + Ov-NC. GAB1, GRB2-associated binding 1; PTK6, protein tyrosine kinase 6; RT-qPCR, reverse transcription-quantitative PCR; Ov, overexpression; NC, negative control; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling.
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