Journal: PLOS Pathogens
Article Title: The splicing factor kinase, SR protein kinase 1 (SRPK1) is essential for late events in the human papillomavirus life cycle
doi: 10.1371/journal.ppat.1012697
Figure Lengend Snippet: (A) Diagram of the early region of the HPV16 genome downstream of the E6E7 gene region. P 670 , HPV16 late promoter located at nucleotide 670 in the E7 coding region. p(A)E, the HPV16 early polyadenylation site. Open rectangles, HPV16 early genes E1, E2, E4, E5. Below is the genome map is shown a diagram of the major spliced mRNA encoding E2. Shaded rectangles, open reading frames. Dotted lines represent RNA sequences spliced out to form the E2 mRNA. AAAn, poly(A) tail. Black chevrons indicate forward and reverse primers used in the first round of the nested RT-PCR reaction shown in (B). A red split chevron indicates the forward primer used in the second round of the nested PCR amplification shown in (B). This cross-splice epitope primer was used with the reverse primer located inside the E2 open reading frame. SD880, splice donor site at nucleotide 880. SA2709, splice acceptor site at nucleotide 2709. (B) Upper panel: ethidium bromide-stained gel showing the products of an RT-PCR reaction to amplify the E2 mRNA from NIKS16 cells differentiated in monolayer and treated with DMSO (lane 1) or with SRPIN349 (lane 2), not inhibitory to SRPK1, or with SRPIN340 (lane 3). Lower panel: GAPDH cDNA was amplified as a loading control. The gel picture is split because the GAPDH reactions were electrophoresed on the right-hand side of the same gel used to visualise the E2 amplification products. These were electrophoresed on the left-hand side adjacent to the size markers. (C) Western blot showing levels of E2 in monolayer-cultured and partially differentiated NIKS16 cells (differentiated for six days) mock-treated, DMSO-treated or treated with either 10 µM or 50 µM SRPIN340 for 48 hours. Protein extract from HPV-negative differentiated monolayer-cultured NIKS cells was included in the experiment to demonstrate specificity of the E2 antibody. β-tubulin was used as a loading control. (D) Graph showing quantification of E2 levels relative to the β-tubulin loading controls in the various experimental conditions used in the western blot in (C). The data shown are the mean and standard deviation from the mean from quantification of three separate western blot experiments. p<0.05*, p-value indicating a significant difference in E2 expression levels. (E) Graph showing quantification of E2 levels relative to the β-tubulin loading controls using protein extracts from NIKS16 cells grown in differentiated 3D raft tissue cultures. The data shown are the mean and standard deviation from the mean from three separate western blot experiments Representative western blots showing E2 and β-tubulin levels are shown above the graph. p<0.05*, p-value indicating a significant difference in E2 expression levels. (F) Immunofluorescence staining with an E2 antibody (green staining, left hand panels) and counterstained with DAPI (blue staining, right hand panels) of sections of differentiated 3D cultured NIKS16 raft tissues topically treated with DMSO or with 10 µM SPRIN340. Dotted lines indicate the basal membrane of the tissues. Size bars=50 µm.
Article Snippet: Membranes were incubated with primary antibody diluted in PBS-T (PBS with 0.1% Tween) containing 5% FBS overnight at 4 o C before being washed three times with PBS-T. Primary antibodies were SRSF1 (1:1000, Mab96, Thermo Fisher Scientific, catalogue # 32-4500), SRSF2 (1:1000, Abcam, catalogue #Ab204916), SRSF3 (1:500, Life Technologies, UK, catalogue #334200), SRPK1 (1:500, clone G211-637 BD Transduction Laboratories, catalogue #611072), α-tubulin (1:5000, clone A11126, Thermo Fisher Scientific catalogue # 236-10501), involucrin (1:1000 clone SY5, Merck, UK, catalogue #19018), GAPDH (1:1000, Meridian Life Sciences, UK, catalogue #H86504M, clone 6C5), HPV16 E2 (1:500 Santa Cruz, Germany, catalogue #sc-53327, TVG271), HPV16 E6 (1:500, Santa Cruz, Germany, catalogue # sc-365089), HPV16 E7 (1:500 Santa Cruz, Germany, catalogue #sc-51951), HPV16 L1 (1:500 Dako, Denmark, clone K1H8), p53 (1:700, BD Pharmingen, UK, catalogue #554294), Rb (1:500, Cell Signaling Technologies, Germany, catalogue #9309, clone 4H1), MCM2 (1:500, Abcam, UK, catalogue #Ab133325), Ki67 (1:1000, Abcam, UK, catalogue #Ab197234), keratin 10 (1:500, Abcam, UK, catalogue #Ab9025).
Techniques: Reverse Transcription Polymerase Chain Reaction, Nested PCR, Amplification, Staining, Control, Western Blot, Cell Culture, Standard Deviation, Expressing, Immunofluorescence, Membrane
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