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. 2011 Dec 7;21(11):2058–2070. doi: 10.1089/scd.2011.0505

FIG. 2.

FIG. 2.

Involvement of integrin β1/FAK/Src and laminin receptor-1 (LR-1)/PKC on Cx43 phosphorylation (A) Cells were treated with integrin β1 neutralizing antibody (Ab, 1 μg/mL) or IgG (negative control; 1 μg/mL) for 30 min before laminin-111 treatment for 30 min, and expression of p-FAK or p-Src was detected by western blot. The lower panel depicts the mean±SD of four independent experiments for each condition as determined from densitometry relative to FAK or Src. *P<0.05 versus control of p-FAK; **P<0.05 versus laminin-111 of p-FAK; #P<0.05 versus control of p-Src; ##P<0.05 versus laminin-111 of p-Src. (B) Cells were treated with protein phosphatase 2 (PP2, FAK/Src inhibitor; 10–6 M) for 30 min before laminin-111 treatment for 30 min, and the expression of p-Cx43 was detected by western blot. The lower panel depicts the mean±SD of three independent experiments for each condition as determined from densitometry relative to β-actin. *P<0.05 versus control; **P<0.05 versus laminin-111. (C) Cells were treated with integrin β1 neutralizing Ab, PP2, and IgG for 30 min before laminin-111 treatment for 24 h, harvested, and subjected to propidium iodide (PI) staining for cell-cycle analysis by flow cytometry. Gates were configured manually to determine the percentage of cells in the S phase based on DNA content. Data are calculated using proliferation indices [(S+G2/M)/(G0/G1+S+G2/M)]×100 and reported as mean±SD of three independent experiments, each conducted in triplicate. *P<0.05 versus control;. **P<0.05 versus laminin-111. (D) Cells were treated with laminin-111 for 30 min, separated into cytosol and plasma membrane fractions, and the expression of PKCγ, PKCδ, or PKCζ protein detected by western blot. (E) Cells were treated with LR-1 neutralizing Ab (1 μg/mL) or IgG for 30 min before laminin-111 treatment for 30 min, and the expression of p-PKC was detected by western blot. The lower panel depicts the mean±SD of four independent experiments for each condition as determined from densitometry relative to PKC. *P<0.05 versus control; **P<0.05 versus laminin-111. (F) Cells were treated with bisindolylmaleimide I (PKC inhibitor; 10–6 M) for 30 min before laminin-111 treatment for 30 min, and the expression of p-Cx43 was detected by western blot. The lower panel depicts the mean±SD of three independent experiments for each condition as determined from densitometry relative to β-actin. *P<0.05 versus control; **P<0.05 versus laminin-111. (G) Cells were treated with LR-1 neutralizing Ab, bisindolylmaleimide I, or IgG for 30 min before laminin-111 treatment for 24 h, harvested, and subjected to propidium iodide (PI) staining for cell-cycle analysis by flow cytometry. Gates were configured manually to determine the percentage of cells in the S phase based on DNA content. Data are calculated using proliferation indices [(S+G2/M)/(G0/G1+S+G2/M)]×100 and reported as mean±SD of three independent experiments, each conducted in triplicate. *P<0.05 versus control;. **P<0.05 versus laminin-111. FAK, focal adhesion kinase.