endogenous pc2  (Santa Cruz Biotechnology)

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 1. Schematic representation of interaction between various PC2 and a-actinin segments revealed by yeast two-hybrid system. Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘ þ ’ and ‘ 2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Construct, Library Screening, Residue

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 2. Interaction between various PC2 segments and a-actinins by GST pull-down assay. (A) E. coli extracts expressing GST-tagged PC2 polypeptides PC2N, PC2C, PC2CA, PC2CB, PC2CC, PC2CD or GST alone or containing only the binding buffer were visualized by the GST antibody. (B and C) Fusion proteins were incubated with purified non-muscle a-actinin (NM ACTN) from chicken gizzard (B) and muscle a-actinin protein (M ACTN) from rabbit skeletal muscle (C). Glutathione–agarose beads were used to pre- cipitate GST epitope binding proteins. The resultant protein samples were immunoblotted with a-actinin antibodies BM75.2 (non-muscle) or EA53 (muscle). The molecular mass markers (in kDa) are shown.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Pull Down Assay, Expressing, Binding Assay, Incubation

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 3. Binding of PC2 with a-actinins by dot blot overlay assay. Purified non-muscle or muscle a-actinin were spotted on nitrocellulose membranes, incubated with total protein lysates of HEK293 and MDCK cells in a blocking buffer containing 1 mM Ca2þ or 1 mM EGTA and PBS buffer (Control). After washes, the membranes were immunoblotted by 1A11. PC2 signals were detected on dots spotted with chicken gizzard non-muscle a-actinin, rabbit skeletal muscle a-actinin or PC2C (positive control), but not on negative con- trols spotted with bovine albumin (BSA) or PBS binding buffer.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Binding Assay, Dot Blot, Overlay Assay, Incubation, Blocking Assay, Control, Positive Control

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 4. Characterization of polycstin-2 antibodies G20 and 1A11. (A) Left panel, total protein from MDCK cells was precipitated with goat G20, non-immune goat IgG (2G20), mouse 1A11 or non-immune mouse IgG (21A11) and detected with 1A11. Right panel, the same membrane was stripped off and re-probed with 1A11 after incubation with the fusion protein GST–PC2C as the blocking peptide. (B) Precipitated samples in panel A were detected with G20 (left panel) or with G20 following pre-incubation with its antigen peptide (right panel). (C and D) MDCK cells stably expressing GFP–PC2 were stained with G20 (red) or visualized for GFP (green, no staining), in the absence (C) and in the presence (D) of the blocking peptide. (E) Ciliated MDCK cells (5 days post-confluency) were co-stained for acetylated a-tubulin and for PC2 (with G20).

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Membrane, Incubation, Blocking Assay, Stable Transfection, Expressing, Staining

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 5. Association between endogenous PC2 and a-actinins in HEK293 and MDCK cells and rat kidney and heart tissues. (A) Total protein from rat heart was precipitated with either muscle a-actinin antibody EA53 or non-immune mouse IgG (2EA53) and detected with antibody 1A11. (B) Reciprocal to A, total protein from rat heart was precipitated with 1A11 antibody or non-immune mouse IgG and probed with EA53. Total cell lysates from HEK293 (C) and MDCK cells (E) and total protein from rat kidney (G) were precipitated with non-muscle a-actinin antibody BM75.2 or non-immune mouse IgG (2). The precipitates were detected with1A11. In reciprocal co-IP experiments, cell lysates from HEK293 (D) and MDCK cells (F) and total protein from rat kidney (H) were pre- cipitated with 1A11 or non-immune mouse IgG (2). The precipitates were detected with BM75.2. Total protein from rat kidney and heart (I and J) was pre- cipitated with 1A11or non-immune mouse IgG (21A11). These precipitates and the total lysates from rat kidney and heart were detected by the spectrin antibody.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Co-Immunoprecipitation Assay

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 6. Subcellular co-localization of PC2 and a-actinin in NIH 3T3, MDCK and IMCD cells and hST vesicles. (A) Localization of endogenous non-muscle a-actinin and transiently transfected PC2 in 3T3 cells, revealed using IF with antibodies BM75.2 and G20, respectively. (B and C) Localization of endogenous non-muscle a-actinin and PC2 in subconfluent MDCK (B) and IMCD (C) cells. Lower panels are expanded pictures of cells from within the square boxes shown in B3 and C3 upper panels. Triangles and arrows in B3 and C3 lower panels indicate the plasma membrane and cell–cell junction localizations, respectively. (D) Cell surface biotinylation in MDCK and IMCD cells. Cells were first labeled with Sulfo-NHS-biotin and precipitated with avidin beads (upper panel, Surface). The remaining flow-through samples were then precipitated with 1A11 and protein G beads and detected by 1A11 in western blotting. Total lysates were also loaded as shown. IMCD cell extracts (Lysate, Surface and Flow-thru without enrichment) were also detected for Naþ/Kþ ATPase a1 subunit, as a plasma mem- brane marker (positive control), and calreticulin, as an endoplasmic reticulum marker (negative control) (lower panels). (E) Interaction and distribution of endogenous non-muscle a-actinin and PC2 determined in hST apical membrane vesicles in the absence and presence of cytochalasin D (CD) (10 mg/ml for 24 h) treatment. For co-IP, vesicles were lysed with CellLytic-M buffer, precipitated with BM75.2 and detected by 1A11. Total lysates were loaded on the left two lanes to indicate the PC2 protein levels. For IF, vesicles were co-stained with goat anti-PC2 G20 (green) and mouse anti-a-actinin BM75.2 (red). Repre- sentative merged images (green plus red) with and without cytochalasin D treatment were shown. Horizontal bars ¼ 20 mm.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Transfection, Clinical Proteomics, Membrane, Labeling, Avidin-Biotin Assay, Western Blot, Marker, Positive Control, Negative Control, Co-Immunoprecipitation Assay, Staining

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 7. Effect of a-actinin on in vitro translated PC2 channel activity in a lipid bilayer system. (A) Representative tracings of in vitro translated, reconstituted PC2 at þ40 mV before and after addition of 250 nM (final concentration) of non-muscle a-actinin to the cis chamber. Cis chamber, 150 mM Kþ; trans chamber, 15 mM Kþ (Materials and Methods). Tracings at sites (a–c) are shown below with an expanded scale. (B) Representative tracings recorded at þ40 and þ60 mV before and after a-actinin addition. (C) Averaged PC2 single-channel currents recorded at þ40 mV before (N ¼ 7) and after (N ¼ 6, paired) addition of a-actinin.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: In Vitro, Activity Assay, Concentration Assay

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 8. Effect of a-actinin on the current amplitude and subconductance states of in vitro translated PC2 channel in a lipid bilayer system. (A) Representative single-channel tracings and corresponding all-point histograms (below each tracing) indicating the increase in single-channel conductance after the addition of non-muscle a-actinin. Data were obtained at þ40 mV. (B) Current–voltage relationship of the largest single-channel conductance in PC2. Dashed and solid lines correspond to the control condition (no a-actinin, as previously reported) (16) and the presence of a-actinin (N ¼ 5), respectively.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: In Vitro, Control

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 9. Regulation of hST PC2 channel function by a-actinin. (A) Representative single-channel tracing (left panels) at þ40 mV of reconstituted hST apical membranes in asymmetrical KCl solutions (Materials and Methods). The corresponding all-point histograms are shown on the right panels. Upper panels, in the absence of a-actinin; lower panels, in the presence of non-muscle a-actinin in the cis chamber. (B) Upper left panel, representative tracings recording at þ40 mV before and after the addition of a-actinin (as indicated). Lower left panel, averaged tracing from six experiments. Right panel, channel open probability (NPo) averaged from the six experiments before and after the application of a-actinin. (C) Cation channel activity in the presence of trans amiloride (100 mM) averaged from six membrane patches, under the same ionic conditions as in (A) and (B) and at þ40 mV. This is compared with channel activity in the absence of amiloride averaged from eight patches of membrane (P , 0.0002).

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Activity Assay, Membrane