Fig 1: NOTCH2 was knocked down using shRNA. The expression of NOTCH2 mRNA and Notch 2 protein in Hep-2 cells 48 h after shRNA transfection measured by (A) reverse transcription-quantitative polymerase chain reaction, and (B) western blot with densitometric analysis, respectively. Notch 2-shRNA samples demonstrate a significantly reduced level of Notch 2. The data represent the results of three independent experiments. *P<0.05 vs. non-transfected samples, #P<0.05 vs. negative-shRNA samples. No significant difference was observed between the non-transfected and the negative-shRNA samples (P>0.05). shRNA, short hairpin RNA.
Fig 2: Knockdown of NOTCH2 inhibits the migration and invasion of Hep-2 cells. (A) NOTCH2 shRNA transfected Hep-2 cells exhibited inhibited cellular motility compared with the control cells. (B) The number of cells that migrated through uncoated filters (no Matrigel) represents the migratory ability of Hep-2 cells. (C) Representative images of the Transwell assay without Matrigel (upper panel) or pre-coated with Matrigel (lower panel) following shRNA transfection. (D) The number of cells that were able to pass through filters pre-coated with Matrigel represents the invasive ability of Hep-2 cells. The cell counts are presented as the mean ± standard deviation of =5 randomly selected low-power fields (×200) from three independent experiments. *P<0.05 vs. non-transfected samples, #P<0.05 vs. negative-shRNA samples. shRNA, short hairpin RNA.
Fig 3: Knockdown of NOTCH2 affects the expression of Notch 2 signaling pathway target genes in Hep-2 cells. (A) Western blot analysis demonstrating that the expression levels of p-ERK, c-Myc and Bcl-2 were downregulated, and the expression of Bax was upregulated. No differences were observed in the expression of t-ERK, p-Akt and t-Akt. (B) Quantification of the protein bands by densitometry. The results of three independent experiments are presented. All the histograms present the GAPDH-normalized mean ± standard deviation of the band density from the three experiments. *P<0.05 vs. non-transfected cells, #P<0.05 vs. negative shRNA. shRNA, short hairpin RNA; p, phospho; t, total; ERK, mitogen-activated protein kinase; AKT, v-akt murine thymoma viral oncogene homolog 1; c-Myc, v-myc avian myelocytomatosis viral oncogene homolog; Bax, BCL2-associated X protein; Bcl2, B-cell CLL/lymphoma 2.
Fig 4: Effect of flutamide (F) and EDS on Notch1 and Notch2 expression in peripubertal rat testis. (A, B) Relative expression of Notch1 and Notch2 mRNAs was determined using real-time RT-PCR analysis. The histograms are the quantitative representation of data of three independent analyses (n = 6 each group). The expression values of the individual genes were normalized to the mean expression of the reference genes (Rn18s, B2m and Actb) as an internal control. Relative quantification (RQ) is expressed as mean ± SD. Significant differences from control values are denoted as **p < 0.01 and ***p < 0.001. (A’, B') Relative protein levels of N1ICD and N2ICD were determined using western blot. The histograms are the quantitative representation after densitometry of data (mean ± SD) of three independent analyses (n = 6 each group). The relative level of studied protein was normalized against its corresponding actin data point. The protein levels within the control group were arbitrarily set as 1. Significant differences from control values are denoted as *p < 0.05, **p < 0.01, and ***p < 0.001
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