Involucrin sy5

Total protein was resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for Phospho-STAT3 (S727) (ab32143, abcam), Phospho-STAT3 (Y705) (9131, Cell Signalling Technology (CST)), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology), Phospho-STAT5 (Y694) (9314, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), involucrin (SY5, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (C1P5, Santa Cruz Biotechnology), HPV 16 E7 (ED17, Santa Cruz Biotechnology), Phospho-ERK1/2 (Thr202/Tyr204) (43705, CST), Phospho-JNK (Thr183/Tyr185) 4668, CST), Phospho-p38 (Thr180/Tyr182) (9211, CST), Bcl xL (H-62, Santa Cruz Biotechnology), Cyclin D1 (A-12, Santa Cruz Biotechnology) p53 (FL-393, Santa Cruz Biotechnology), p21 (2947, CST), FLAG (F3165, Sigma), GFP (B-2: sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology). Western blots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL (Thermo/Pierce).

Cells were fixed, and prepared for immunocytochemistry staining following the protocol in Jackson et al.^{17 (link)}. Antibodies and concentrations are listed in Table 2. Images were captured at random positions using an inverted epi-fluorescence microscope (Nikon Eclipse Ti with a DS-Qi1 camera; Nikon Instruments, Tokyo, Japan) at 200X magnification. The exposure length and gain were kept constant. ImageJ software was used to process the images. The percentage of positive staining for each marker was calculated based on an average from counting ~100 cells from randomly selected positions in n = 4 wells. Counts were verified by two independent investigators.Table 2

Antibodies Used in Immunocytochemistry.

Antibody	Epitope	Supplier	Concentration
Proliferating Cell Nuclear Antigen (PCNA)	PC10 (mouse)	Dako	1:500
Cleaved Caspase-3	D175 (rabbit)	Cell Signaling	1:400
Tumor Protein P63 (P63)	EPR5701 (rabbit)	Abcam	1:300
Tumor Protein P63 (P63)	4A4 (mouse)	Abcam	1:100
ABC Transporter Family G2 (ABCG2)	BXP21 (mouse)	Santa Cruz	1:100
CCAAT/enhancer-binding protein delta (C/EBPδ)	Polyclonal (rabbit)	Abcam	1:600
Cytokeratin 14 (CK14)	LL002 (mouse)	Abcam	1:300
Cytokeratin 10 (CK10)	Polyclonal (rabbit)	Abcam	1:800
Involucrin	SY5 (mouse)	Santa Cruz	1:300

Keratinocytes containing wild type HPV18 genomes or the mutant E6ΔPDZ genome were grown in organotypic raft cultures by seeding the keratinocytes onto collagen beds containing J2-3T3 fibroblasts [13 (link)]. Once confluent the collagen beds were transferred onto metal grids and fed from below with FCS-containing E media without EGF. The cells were allowed to stratify for 14 days before fixing with 4% formaldehyde. The rafts were paraffin-embedded and 4 μm tissue sections prepared (Propath UK, Ltd., Hereford, UK).
For analysis of Phospho-STAT3 (S727) (ab32143, abcam), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology and 9132, CST), involucrin (SY5, Santa Cruz Biotechnology) and HPV18 E1^E4 (mouse monoclonal antibody 1D11 [84 ]) expression, the formaldehyde-fixed raft sections were treated with the sodium citrate method of antigen retrieval. Briefly, sections were boiled in 10 mM sodium citrate with 0.05% Tween-20 for 10 minutes. Sections were incubated with appropriate antibodies and immune complexes visualized by using Alexa 488 and 594 secondary antibodies (Invitrogen). The nuclei were counterstained with the DNA stain 4’,6-diamidino-2-phenylindole (DAPI) and mounted in Prolong Gold (Invitrogen).