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. 2020 Jan 8;69(4):736–748. doi: 10.2337/db19-0713

Figure 4.

Figure 4

Depletion of p110δ attenuates Akt activation induced by VEGF, bFGF, or EGF. A: Schematic of the human genomic PIK3CD locus. One target for generating its sgRNA was selected at the human genomic PIK3CD exon 4 and named as PK2 (green line). The PAM sequences are marked in a blue line. B: Genomic DNA isolated from HRECs with CRISPR/Cas9 targeting to PIK3CD by sgRNA-PK2 or control by sgRNA-lacZ was used to PCR amplify the DNA fragments around the sgRNA-PK2 protospacer with primers P24F1 (5′-GGGAGGTTTGGACCCCCAG-3′) and P24R1 (5′-ACCTTTGCCGATGAGGAGGC-3′) for Sanger DNA sequencing. C: Lysates of HRECs expressing sgRNA-lacZ or PK2/Cas9 were subjected to Western blot analysis using the indicated antibodies. This is representative of three independent experiments. D–H: The characterized HRECs in B were cultured to 90% confluence and then starved of growth factors for 8 h. Subsequently, these cells were treated with VEGF, bFGF, and EGF (10 ng/mL) for 15 min, and their clarified lysates were subjected to Western blot analysis using the indicated antibodies. A representative of three independent experiments is shown. The intensity of the bands from three experiments was quantified as dot plots. Data in F and H are mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by the Tukey post hoc test.