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. 2009 May 4;29(13):3582–3596. doi: 10.1128/MCB.01417-08

FIG. 7.

FIG. 7.

Mutation of either LKB1 S307 or S428 to alanine inhibits LKB1 binding to STRAD and CRM1. (A) A549 cells were transfected with WT LKB1 or mutant LKB1 S307A, LKB1 S428A, LKB1 D194A, or LKB1 SL26. STRADα or LKB1 was immunoprecipitated (IP) using a specific antibody, and LKB1 or STRADα was detected by Western analysis. The blot is representative of four blots from four individual experiments (n = 4). ♣, P < 0.05 versus WT LKB1 or mutant LKB1 S307A, LKB1 S428A, or D194A. IgG, immunoglobulin G. (B) Mutation of S307 and S428 reduced the ONOO−-enhanced association of LKB1 with STRADα. A549 cells were transfected with WT LKB1 or mutant LKB1 S307A, LKB1 S428A, or LKB1 S307A S428A and treated with ONOO− (50 μM) for 15 min. (Upper panel) Protein levels of LKB1, MO25α, and STRADα in total cell lysates were detected by Western blot analysis. (Bottom panel) STRADα was immunoprecipitated using a specific antibody, and LKB1 was detected by Western analysis. The blot is representative of four blots from four individual experiments (n = 4). (C and D) HUVECs were pretreated with LMB (200 nM) for 1 h followed by treatment with ONOO− (50 μM) for 15 min; subcellular fractions were prepared as described in Materials and Methods. The protein levels of LKB1 (C) and CRM1 (D) in each fraction were analyzed by Western blotting. β-Actin was used as the loading control for the cytoplasmic fraction, and histone H2AX was used as the loading control for the nuclear fraction. The blot is a representative of three blots from three individual experiments (n = 3). ♣, P < 0.05 versus control; †, P < 0.05 versus ONOO−. (E) Confluent HUVECs were pretreated with LMB followed by ONOO− treatment; cell lysates were prepared as described in Materials and Methods. Phosphorylation of AMPK at T172 was detected by Western blotting. The blot is a representative of three blots from three individual experiments (n = 3). ♣, P < 0.05 versus control; †, P < 0.05 versus ONOO−. (F and G) A549 cells transfected with WT LKB1 and LKB1 mutants were treated with ONOO− (50 μM) for 15 min. (F) LKB1 was immunoprecipitated, and CRM1 and STRADα were detected by Western blotting. (G) STRADα was immunoprecipitated with a specific antibody, and CRM1 was detected by Western analysis. The blot is a representative of four blots from four individual experiments (n = 4).