FIGURE 7:

Ltn1 activity regulates RSK1/2 signaling. (A) 293T cells were transfected with control siRNA oligos (siC) or three separate Ltn1-targeting siRNA oligos. Two days after transfection, cells were serum starved overnight and were then untreated or treated with 1 μM PMA for 15 min. Whole-cell extracts were immunoblotted as indicated. (B) 293T cells were transfected with control siRNA oligos (siC) or three separate Ltn1-targeting siRNA oligos. Two days after transfection, cells were serum starved overnight and were then treated with 100 nM Torin 1 or 10 μM RSK inhibitor (RSKi) as indicated for 1 h prior to PMA treatment for 15 min. Whole-cell extracts were immunoblotted as indicated. (C) The band intensities from immunoblots from replicate experiments in 293T cells where Ltn1 was knocked down with individual siRNA oligos and the levels of pTSC2 (S1798) and total TSC2 were measured on serum starvation and subsequent PMA addition were quantified using Image Lab software volume tools (Bio-RAD). All immunoblots were imaged using a ChemiDoc XRS+ system which uses a charged-coupled device (CCD) detection system. A ratio of the background corrected band intensities of pTSC2:TSC2 was generated for each siRNA oligo targeting Ltn1 and normalized to the ratio observed in siControl-treated cells. Error bars represent SD (N = 5 for siLtn1 oligo 1 and 3, N = 4 for siLtn1 oligo 2). (D) 293T cells were transfected with control siRNA oligos (siC) or three separate oligos targeting Ltn1 or NEMF. Two days after transfection, cells were serum starved overnight and were then untreated or treated with 1 μM PMA for 15 min. Whole-cell extracts were immunoblotted as indicated. (E) 293 Flp-In cells with dox-induced expression of BirA*-FLAG-NEMF were transfected with control scrambled siRNA oligos (siC) or NEMF-targeting siRNA oligos. Forty-eight hours after siRNA transfection, BirA*-FLAG-NEMF expression was induced with dox for 16 h before cells were harvested. Whole-cell extracts were immunoblotted as indicated.