Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Representative Western blot for PI(4,5)P 2 in total cell extracts of RM+ N/TERTs and KGM N/TERTs. Equal protein loading was confirmed by immunoblotting for Tubulin. ( B ) Representative Western blot for PI(4,5)P 2 in total cell extracts of KGM N/TERT grown in low calcium and then switched to high calcium concentrations for up to 8 days. Calcium induced keratinocyte differentiation was confirmed by Western blotting for Loricrin. Equal protein loading was confirmed by immunoblotting for tubulin. ( C ) Representative Western blot for PI(4,5)P 2 in total cell extracts of empty vector positive KGM N/TERT-pLXSN or KGM N/TERT-8E6. Difference in intensity of PI(4,5)P 2 bands in KGM N/TERTs between Figure and is due to 80 μg total protein and long exposure of blot to film versus 40 μg total protein and short exposure. Equal protein loading was confirmed by immunoblotting for tubulin. ( D ) Representative Western blot for PI(4,5)P 2 in total cell extracts of KGM N/TERT-pLXSN and KGM N/TERT-8E6 treated with skimmed milk containing either PI(4,5)P 2 specific antibodies (top) or in milk containing100 mM Neomycin (bottom). ( E ) Total cell extracts of KGM N/TERT-8E6 were treated with 0, 5, 10 or 20 mU Phospholipase C (PLC). ( F ) Quantification of phosphoinositide levels in HPV8-E6 expressing keratinocytes. Relative amounts of PIP and PIP 2 levels in KGM N/TERT-pLXSN and KGM N/TERT-8E6 cells. Bars present phosphoinositide values normalized to phosphatidylcholine (PC) levels (PIP x /PC). Data are presented as mean ± SD ( n = 3). ( G – H ) Effect of E6 expression on the relative distribution of PIP ( G ) and PIP 2 ( H ) molecular species. Lipid species are annotated based on their fatty acyl composition x:y, with x = total number of C atoms in both fatty acyl chains, and y = total number of double bonds in both fatty acyl chains). Data are normalized to 100% within each lipid class and are displayed as mean ± SD ( n = 3).

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Western Blot, Plasmid Preparation, Expressing

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Representative immunocytochemical staining of PI(4,5)P 2 in KGM N/TERT-pLXSN and KGM N/TERT-8E6 showing an increase in nuclear PI(4,5)P 2 levels in HPV8-E6 positive cells (blue: DAPI; green: PI(4,5)P 2 ). ( B ) Representative immunofluorescence staining image of PI(4,5)P 2 on organotypic skin cultures based on de-epidermalized human dermis as matrix, which was repopulated with wt PHK or PHK coding for the empty retroviral vector pLXSN or pLXSN-8E6. The organotypic cultures were grown for 14 days at the air-liquid interphase, followed by fixing and embedding in paraffin.

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Staining, Immunofluorescence, Plasmid Preparation

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: Skin biopsies were taken from FVB/n-wt ( n = 4) and K14-HPV8-E6 ( n = 4) mice out of the UV irradiated skin area 13 days or 24 days following UV treatment. Additionally, skin biopsies were taken from non-irradiated ( n = 4) FVB/n-wt and K14-HPV8-E6 ( n = 4) mice, respectively. Paraffin sections were stained for PI(4,5)P 2 (blue: DAPI; green: PI(4,5)P 2 ; dashed line: basement membrane zone; d: dermis; e: epidermis). The magnified image of wt skin 24 days following UV treatment demonstrates unspecific staining in the cornified cell layer.

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Irradiation, Staining

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Representative immunofluorescence staining for PI(4,5)P 2 in normal human skin, skin SCC of the normal population (SCC/NP) and HPV8 positive EV-SCC. Histology of the EV lesions is shown by HE staining (blue: DAPI; green: PI(4,5)P 2 ; dashed line: basement-membrane zone; d: dermis; e: epidermis). ( B ) Representative immunofluorescence staining for PI(4,5)P 2 in malignant melanoma (MM, n = 5), Merkel cell polyomavirus positive Merkel cell carcinoma (MCC, n = 5) and basal cell carcinoma (BCC, n = 5) (blue: DAPI; green: PI(4,5)P 2 ; dashed line: basement-membrane zone; d: dermis; e: epidermis).

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: Representative immunofluorescence staining for PI(4,5)P 2 in normal cervix ( n = 3), and HPV16 positive CIN I ( n = 3), CIN II ( n = 5), CIN III ( n = 11) and cervical cancer (CC, n = 3) (blue: DAPI; green: PI(4,5)P 2 ). Histology of the tissue is shown in the HE staining images.

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Extracts from C33a cells, transiently transfected with either empty vectors or plasmids coding for Flag-8E6-wt, -P31S, -P38S, -F41Y, -W63T, -L61A/W63A, -V68A, -E83A, -D96A, -D126A, -K136N, -K149A were incubated with M2-FLAG-agarose. Co-immunoprecipitated of MAML1 and 10% of the input extracts were subjected to Western blots with specific antibodies. Expression of HPV8-E6 was confirmed by a Western blot for the Flag-tagged HPV8-E6 protein. Equal protein loading was confirmed by immunoblotting for tubulin. ( B ) N/TERTs were transduced with retroviruses encoding for either the empty vector pLXSN-HA or HA-tagged HPV8-E6wt, -L61A, -W63T or L61A/W63A. PI(4,5)P 2 was detected with specific antibodies. Expression of HPV8-E6 was confirmed by Co-IP of the HA-tagged proteins. Equal protein loading was confirmed by immunoblotting for tubulin.

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Transduction, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Quantification of PIP4KIIα, β, γ and PIP5KIα, β, γ mRNA expression by RT-qPCR in KGM N/TERTs and RM+ N/TERTs ( n = 3 independent experiments measured in duplicates). Data were normalized to the HPRT1 mRNA levels and are presented as mean ± SEM. The relative gene expression level of RM+ N/TERTs was set as 1 ( ** p < 0.01; *** p < 0.001; nq: not quantifiable; ns: not significant). ( B ) Quantification of PIP4KIIα, β, γ and PIP5KIα, β, γ mRNA expression by RT-qPCR in KGM N/TERT-pLXSN and KGM N/TERT-8E6 ( n = 3 independent experiments measured in duplicates). Data were normalized to HPRT1 mRNA levels and are presented as mean ± SEM. The relative gene expression level of KGM N/TERT-pLXSN was set as 1 ( * p < 0.05; ** p < 0.01; nq: not quantifiable). ( C ) Extracts from C33a cells, transiently transfected with expression vectors for empty vector or Flag-HPV8-E6wt were incubated with M2-FLAG-agarose. Co-immunoprecipitated OCRL-1 and 10% of the input extracts were detected by Western blot with specific antibodies ( n = 5). The expression of HPV8-E6 was confirmed by a Western blot against the Flag tag. Equal protein loading was confirmed by immunoblotting for tubulin. ( D ) Schematic diagram of the nitrocellulose membrane with immobilized phospholipids in 100 picomole spots (PIP-strip; left image); LPA: lysophosphatidic acid; LPC: lysophosphocholine; PI: phosphatidylinositol; SIP: sphingosine-1-phosphate; PA: phosphatidic acid. Representative images of PIP-strips incubated with total cell extracts of C33a cells transiently transfected with pXJ41-Flag (middle image) or pXJ41-8E6-Flag (right image). Membranes were washed and lipid-bound Flag-8E6 was detected with monoclonal anti-Flag antibody. ( E ) The left image shows the PI(4,5)P 2 pathway flow in a normal keratinocyte. The right image schematically summarizes HPV8-E6 targets involved in PI(4,5)P 2 metabolism. The figure also shows that the PIP5K pathway is the major and PIP4K the minor route of PI(4,5)P 2 synthesis.

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, FLAG-tag, Stripping Membranes

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: Extracts from KGM N/TERT-pLXSN and KGM N/TERT-8E6 were incubated with A/G-agarose coupled with either CAND1, SND1 or normal IgG-mouse antibodies. Co-immunopecipitated PI(4,5)P 2 and 10% of the input extracts were detected by Western blot with specific antibodies against PI(4,5)P 2 . Total levels of PI(4,5)P 2 , CAND1 and SND1 were detected using specific antibodies. Equal protein loading was confirmed by immunoblotting for tubulin.

Article Snippet: Incubation with primary antibody against PI(4,5)P 2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P 2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT.

Techniques: Incubation, Western Blot

Journal: Nature Communications

Article Title: Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

doi: 10.1038/ncomms6984

Figure Lengend Snippet: ( a , b ) Both syntaxin 1 and syntaxin 4 clusters colocalize with PI(4,5)P 2 . Plasma membrane sheets derived from PC12 cells were immunostained for PI(4,5)P 2 (green), syntaxin 1 ( a ; red) and syntaxin 4 ( b ; red) and imaged by two-colour STED microscopy. The graphs show the fluorescence intensity profiles as indicated in the figures (syntaxin 1 and 4: red profiles; PI(4,5)P 2 : green profiles); yellow bars depict the positions of the domains. ( c , d ) Both PI(4,5)P 2 and cholesterol enhance co-clustering of sx-1TM and sx-4TM. FRET was measured after reconstitution in large unilamellar vesicles containing sx-1TM labelled with Rhodamine Red (donor fluorophore) and sx-4TM with Atto647N (acceptor) composed of porcine brain PC without or with 1 mol% PI(4,5)P 2 and/or 30 mol% cholesterol (± range from two independent reconstitutions, three technical repeats each). ( e ) Clustering of sx-1TM by FRET in the presence or absence of 150 mM or 1 M NaCl and in DOPC liposomes in the absence or presence of 3 mol% PI(4,5)P 2 . ( f ) Clustering of sx-1TM (green) and the sx-1TM oligomerization mutant (purple), monitored by FRET, in C14:1 PC or C18:1 PC liposomes. Error bars: range from two independent reconstitutions, three technical repeats each. Scale bars, 2 μm.

Article Snippet: Antibodies used for immunohistochemistry were syntaxin 1 HPC-1 immunoglobulin-G1 (IgG1, Sigma, clone HPC-1) and rabbit polyclonal antiserum (Synaptic Systems); syntaxin 4 mouse monoclonal IgG1 (Synaptic Systems, clone number 139.2); IgM antibodies against PI(4,5)P 2 (clone Z-A045); mouse monoclonal IgG2a anti-mCherry (Abcam, clone 1C51) and rabbit polyclonal anti-EGFP (Abcam).

Techniques: Derivative Assay, Microscopy, Fluorescence, Mutagenesis

Journal: Nature Communications

Article Title: Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

doi: 10.1038/ncomms6984

Figure Lengend Snippet: ( a ) Two-colour STED microscopy of PC12 cell sheets immunostained for syntaxin 1 and syntaxin 4 shows segregation of endogenous proteins into separate clusters. ( b ) Reduced co-clustering of sx-1TM and sx-4TM in membranes composed of a mixture of PC with different acyl-chain lengths. FRET assay is similar to , but now measuring clustering of TMDs in liposomes composed of either C18:1 PC or an equimolar mixture of C14:1, C16:1, C18:1 and C20:1 PC. All liposomes contained 1 mol% PI(4,5)P 2 and 30 mol% cholesterol (± range from two independent reconstitutions, three technical repeats each). ( c ) Same as in a but now using PC12 cell sheets derived from cells expressing truncation mutants of syntaxin 1 and 4 (sx-1TM; sx-4TM) that are fused to mCherry and EGFP, respectively, and immunostained with antibodies against mCherry and EGFP. ( d ) Control experiment of PC12 cells co-expressing sx-1TM tagged with either mCherry or EGFP, showing co-localization (Scale bar, 2 μm). ( e ) Overlap of clusters in membrane sheets from PC12 cells transfected with various sx-1TM and sx-4TM mutants (all mCherry and EGFP tagged, respectively) measured by determining the Pearson-correlation coefficient. Sx-1FL and sx-4FL; full length constructs of syntaxin 1 and syntaxin 4, respectively (from a ). Each analysis included at least 10 sheets from 3 independent transfections (*** P <0.001, two-sided, unpaired t -test; error bars show s.e.m.). Scale bars, 2 μm.

Article Snippet: Antibodies used for immunohistochemistry were syntaxin 1 HPC-1 immunoglobulin-G1 (IgG1, Sigma, clone HPC-1) and rabbit polyclonal antiserum (Synaptic Systems); syntaxin 4 mouse monoclonal IgG1 (Synaptic Systems, clone number 139.2); IgM antibodies against PI(4,5)P 2 (clone Z-A045); mouse monoclonal IgG2a anti-mCherry (Abcam, clone 1C51) and rabbit polyclonal anti-EGFP (Abcam).

Techniques: Microscopy, Derivative Assay, Expressing, Transfection, Construct

Journal: Nature Communications

Article Title: Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

doi: 10.1038/ncomms6984

Figure Lengend Snippet: Syntaxin membrane clustering is induced by a combination of hydrophobic mismatch (increased by cholesterol-induced membrane thickening) and electrostatic interactions with ions and PI(4,5)P 2 . The membrane clusters are further refined by protein–protein interactions in the aqueous space.

Article Snippet: Antibodies used for immunohistochemistry were syntaxin 1 HPC-1 immunoglobulin-G1 (IgG1, Sigma, clone HPC-1) and rabbit polyclonal antiserum (Synaptic Systems); syntaxin 4 mouse monoclonal IgG1 (Synaptic Systems, clone number 139.2); IgM antibodies against PI(4,5)P 2 (clone Z-A045); mouse monoclonal IgG2a anti-mCherry (Abcam, clone 1C51) and rabbit polyclonal anti-EGFP (Abcam).

Techniques:

Journal: Nature Communications

Article Title: Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

doi: 10.1038/ncomms6984

Figure Lengend Snippet: ( a , b ) Both syntaxin 1 and syntaxin 4 clusters colocalize with PI(4,5)P 2 . Plasma membrane sheets derived from PC12 cells were immunostained for PI(4,5)P 2 (green), syntaxin 1 ( a ; red) and syntaxin 4 ( b ; red) and imaged by two-colour STED microscopy. The graphs show the fluorescence intensity profiles as indicated in the figures (syntaxin 1 and 4: red profiles; PI(4,5)P 2 : green profiles); yellow bars depict the positions of the domains. ( c , d ) Both PI(4,5)P 2 and cholesterol enhance co-clustering of sx-1TM and sx-4TM. FRET was measured after reconstitution in large unilamellar vesicles containing sx-1TM labelled with Rhodamine Red (donor fluorophore) and sx-4TM with Atto647N (acceptor) composed of porcine brain PC without or with 1 mol% PI(4,5)P 2 and/or 30 mol% cholesterol (± range from two independent reconstitutions, three technical repeats each). ( e ) Clustering of sx-1TM by FRET in the presence or absence of 150 mM or 1 M NaCl and in DOPC liposomes in the absence or presence of 3 mol% PI(4,5)P 2 . ( f ) Clustering of sx-1TM (green) and the sx-1TM oligomerization mutant (purple), monitored by FRET, in C14:1 PC or C18:1 PC liposomes. Error bars: range from two independent reconstitutions, three technical repeats each. Scale bars, 2 μm.

Article Snippet: Antibodies used for immunohistochemistry were syntaxin 1 HPC-1 immunoglobulin-G1 (IgG1, Sigma, clone HPC-1) and rabbit polyclonal antiserum (Synaptic Systems); syntaxin 4 mouse monoclonal IgG1 (Synaptic Systems, clone number 139.2); IgM antibodies against PI(4,5)P 2 (clone Z-A045); mouse monoclonal IgG2a anti-mCherry (Abcam, clone 1C51) and rabbit polyclonal anti-EGFP (Abcam).

Techniques: Derivative Assay, Microscopy, Fluorescence, Mutagenesis

Journal: Nature Communications

Article Title: Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

doi: 10.1038/ncomms6984

Figure Lengend Snippet: ( a ) Two-colour STED microscopy of PC12 cell sheets immunostained for syntaxin 1 and syntaxin 4 shows segregation of endogenous proteins into separate clusters. ( b ) Reduced co-clustering of sx-1TM and sx-4TM in membranes composed of a mixture of PC with different acyl-chain lengths. FRET assay is similar to , but now measuring clustering of TMDs in liposomes composed of either C18:1 PC or an equimolar mixture of C14:1, C16:1, C18:1 and C20:1 PC. All liposomes contained 1 mol% PI(4,5)P 2 and 30 mol% cholesterol (± range from two independent reconstitutions, three technical repeats each). ( c ) Same as in a but now using PC12 cell sheets derived from cells expressing truncation mutants of syntaxin 1 and 4 (sx-1TM; sx-4TM) that are fused to mCherry and EGFP, respectively, and immunostained with antibodies against mCherry and EGFP. ( d ) Control experiment of PC12 cells co-expressing sx-1TM tagged with either mCherry or EGFP, showing co-localization (Scale bar, 2 μm). ( e ) Overlap of clusters in membrane sheets from PC12 cells transfected with various sx-1TM and sx-4TM mutants (all mCherry and EGFP tagged, respectively) measured by determining the Pearson-correlation coefficient. Sx-1FL and sx-4FL; full length constructs of syntaxin 1 and syntaxin 4, respectively (from a ). Each analysis included at least 10 sheets from 3 independent transfections (*** P <0.001, two-sided, unpaired t -test; error bars show s.e.m.). Scale bars, 2 μm.

Article Snippet: Antibodies used for immunohistochemistry were syntaxin 1 HPC-1 immunoglobulin-G1 (IgG1, Sigma, clone HPC-1) and rabbit polyclonal antiserum (Synaptic Systems); syntaxin 4 mouse monoclonal IgG1 (Synaptic Systems, clone number 139.2); IgM antibodies against PI(4,5)P 2 (clone Z-A045); mouse monoclonal IgG2a anti-mCherry (Abcam, clone 1C51) and rabbit polyclonal anti-EGFP (Abcam).

Techniques: Microscopy, Derivative Assay, Expressing, Transfection, Construct

Journal: Nature Communications

Article Title: Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

doi: 10.1038/ncomms6984

Figure Lengend Snippet: Syntaxin membrane clustering is induced by a combination of hydrophobic mismatch (increased by cholesterol-induced membrane thickening) and electrostatic interactions with ions and PI(4,5)P 2 . The membrane clusters are further refined by protein–protein interactions in the aqueous space.

Article Snippet: Antibodies used for immunohistochemistry were syntaxin 1 HPC-1 immunoglobulin-G1 (IgG1, Sigma, clone HPC-1) and rabbit polyclonal antiserum (Synaptic Systems); syntaxin 4 mouse monoclonal IgG1 (Synaptic Systems, clone number 139.2); IgM antibodies against PI(4,5)P 2 (clone Z-A045); mouse monoclonal IgG2a anti-mCherry (Abcam, clone 1C51) and rabbit polyclonal anti-EGFP (Abcam).

Techniques:

Journal: Human Molecular Genetics

Article Title: Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids

doi: 10.1093/hmg/ddq271

Figure Lengend Snippet: Interactions of cadherin-23, harmonin and myosin VIIa with phospholipids. ( A ) Binding to non-phosphorylated and phosphorylated forms of PI. Real-time SPR profiles showing the interaction of full-length harmonin-a, myosin VIIa tail or CDH23 + exon68 (1.25 µ m each) with immobilized liposomes containing 20% of PI, PI4P, PI5P or PI(4,5)P 2 . The binding of CDH23 + exon68 and harmonin-a depended strongly on the phosphorylation of the PI lipids, unlike that of myosin VIIa. ( B ) PI(4,5)P 2 -tethered cadherin-23 is able to bind to harmonin-a. Real-time SPR profiles showing the interaction with immobilized 20% PI(4,5)P 2 -containing liposomes of the following proteins (250 n m ): CDH23 + exon68 alone (1, black curve and scale), full-length harmonin-a alone (2, green curve and scale) or CDH23 + exon68 followed by harmonin-a (3, red curve and scale).

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against myosin VIIa ( , ), harmonin-b ( ) or cadherin-23 , mouse monoclonal IgG against Myc (mAb 9E10 from Santa Cruz Biotechnology) and IgM against PI(4,5)P 2 (mAb 2C11 from Molecular Probes, Eugene).

Techniques: Binding Assay

Journal: Human Molecular Genetics

Article Title: Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids

doi: 10.1093/hmg/ddq271

Figure Lengend Snippet: Colocalization of PI(4,5)P 2 with either cadherin-23 or harmonin-a in transfected HeLa cells. Upper panel: CDH23 + exon68 fused to eGFP (eGFP-CDH23 + exon68) and the myc-tagged PH domain of phospholipase C (myc-PH), used as a PI(4,5)P 2 reporter, co-localized at the plasma membrane. Lower panel: full-length harmonin-a fused to eGFP (eGFP-harmonin-a) and myc-PH co-localized at the plasma membrane after H 2 O 2 treatment of the cells. Scale bars: 5 µm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against myosin VIIa ( , ), harmonin-b ( ) or cadherin-23 , mouse monoclonal IgG against Myc (mAb 9E10 from Santa Cruz Biotechnology) and IgM against PI(4,5)P 2 (mAb 2C11 from Molecular Probes, Eugene).

Techniques: Transfection

Journal: Human Molecular Genetics

Article Title: Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids

doi: 10.1093/hmg/ddq271

Figure Lengend Snippet: PI(4,5)P 2 immunodetection in growing (P1) and mature (P15) hair bundles from mouse outer hair cells. Scale bars: 1 µm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against myosin VIIa ( , ), harmonin-b ( ) or cadherin-23 , mouse monoclonal IgG against Myc (mAb 9E10 from Santa Cruz Biotechnology) and IgM against PI(4,5)P 2 (mAb 2C11 from Molecular Probes, Eugene).

Techniques: Immunodetection