Figure 9. Ser⁵² of hnRNP L is hyperphosphorylated in NSCLC cells and regulates the alternative splicing of caspase-9.

(A) A549 and HBEC-3KT cell lines were seeded at the same confluency and in the same culture media 24 hours before IP. IP hnRNP L was resolved by SDS-PAGE and immunoblotted with phospho-specific and hnRNP L antibodies. (B) A549 cells were transfected with WT-hnRNP L (L-WT) (1 μg), Ser⁵²Ala hnRNP L (S52-A) (1 μg), or Ser⁵²Asp hnRNP L (S52-D) (1 μg). Total RNA was analyzed for caspase-9 splice variants. n = 4 from 3 occasions. (C) A549 cells were transfected with WT-hnRNP L, S52-A, or S52-D. Protein lysates were subjected to SDS-PAGE and immunoblotted for myc-tag and β-actin. Empty vector showed no expression of a myc-tagged protein. (D) A549 and HBEC-3KT cell lines seeded at the same confluency and in the same culture media for 24 hours before IP. Cell lines were transfected with WT-hnRNP L (1 μg) or S52-A (1 μg). Ectopically expressed hnRNP L was IP with c-myc tag Ab, resolved by SDS-PAGE, and immunoblotted with anti–phospho-serine and anti–c-myc tag antibodies. (E) Protein lysates were subjected to SDS-PAGE and immunoblot for phospho-Ser⁵² hnRNP L and hnRNP L. Phospho-Ser⁵² antibody for hnRNP L was validated by ELISA, hnRNP shRNA samples, and lack of identifying the Ser⁵²Ala mutant of hnRNP L. (F) Colony formation assays in soft agar. n = 6; error bars represent SEM. *P < 0.005, A549 + S52-A hnRNP L + C9b ectopic versus A549 + S52-A hnRNP L.