Journal of Oncology / 2010 / Article / Fig 5

Research Article

Lysophosphatidic Acid Upregulates Laminin-332 Expression during A431 Cell Colony Dispersal

Figure 5

LPA enhances laminin-332 protein expression in A431 cells. To examine the expression level of Ln-332 chains in LPA-treated A431 cells, cells were serum starved for 24 h and incubated with PBS or 2  πœ‡ M LPA for 12 h. (a, b) Cells were then lysed and 100 πœ‡ g of proteins were separated on an 8% gel under reducing conditions. After separation, the same gel was transferred to a PVDF membrane and blocked with 5% skim milk in 1X TBS and 0.1% Tween 20. After blocking, BM165 (mAb against human laminin 𝛼 3 chain) H-300 (pAb against human laminin 𝛽 3 chain), a pAb against rat laminin 𝛾 2 chain, or a mAb against actin was added at the diluted ratios of 1 : 1000, 1 : 200, 1 : 2000, and 1 : 10000, respectively, and incubated at 4 ∘ C overnight. Anti-rabbit IgG or mouse IgG HRP-conjugated antibody was used as a secondary antibody at the diluted ratio of 1 : 1000. The bands were visualized with an ECL plus system (Perkin Elmer, Waltham, MA). Protein expression levels were quantified using ImageJ. Results showed that LPA treatment consistently increased Ln-332 component expression. Both the Ln-332 𝛼 3 and 𝛽 3 chains were determined to be significantly increased (N = 3, P =  .004), whereas the Ξ³2 chain was only somewhat elevated compared to the PBS control treatment.

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