Cervical cancer cells over-express STAT3 with phosphorylation on both tyrosine and serine residues. (A) Cervical cancer cell lines C33a (HPV-), SiHa & CaSki (HPV16+) and HeLa (HPV18+) (2 × 106 cells) were lysed, and 50 μg of total cellular proteins were resolved on 7.5% SDS-PAGE, electrotransferred to a PVDF membrane, and probed for STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin expression by respective antibodies. (B) STAT3 expression and phosphorylation increases as a function of severity of cervical lesions. Representative immunoblot of STAT3, pSTAT3(Y705), pSTAT3(S727) and β-actin indicating expression of respective protein in total cellular proteins (50 μg) isolated from normal (N), low grade squamous intra-epithelial lesion (LSIL1 & 2), high grade SIL (HSIL) and cervical cancer biopsy tissues (CaCx1, CaCx2) as described in Methods. (C) STAT3 RT-PCR analysis of cDNA derived from cervical cancer cell lines and cervical tissues. Representative ethidium bromide-stained agarose gel (3%) showing specific amplification STAT3 transcripts (318 bp) in the cDNA derived from total RNA of indicated samples (middle panel). Quality and quantity of RNA used for cDNA preparation was examined and confirmed by 1% agarose gel electrophoresis (upper panel). GAPDH RT-PCR (amplicon size 520 bp) was used as internal control (lower panel).