Journal: Scientific Reports

Article Title: Dynamic interplay between sortilin and syndecan-1 contributes to prostate cancer progression

doi: 10.1038/s41598-023-40347-7

Figure Lengend Snippet: Sortilin (SORT) interacts with progranulin (PRGN) and LPL in PCa cells and PRGN is secreted by aggressive PC3 cells. ( a ) Representative confocal images showing co-labelling of cells with anti-PRGN (red) and anti-SORT (green) antibodies. Scale bars; 10 µm. ( b ) Quantification of colocalisation between PRGN and SORT. ( c ) Amount of PRGN in conditioned media (CM) and corresponding cell lysates with quantification of band densities and ratio of PRGN in CM to PRGN in cell lysates. ( d ) Endogenous SORT was immunoprecipitated (IP) with anti-SORT antibodies and PRGN was detected by Western blotting. Input; cell lysate. ( e ) Representative confocal images showing co-labelling of cells with anti-LPL (red) and anti-SORT (green) antibodies. Scale bars; 10 µm. ( f ) Quantification of colocalisation between LPL and SORT. ( g ) Amount of LPL in CM and corresponding cell lysates with quantification of band densities in cell lysates. In ( c ) and ( g ) PNT2 bands from the same membrane were shifted towards LNCaP and PC3 bands. Western blotting signal was normalised using total protein staining. ( h ) Endogenous SORT was immunoprecipitated (IP) with anti-SORT antibodies and LPL was detected by Western blotting. Input; cell lysate. Data are presented as mean ± SD, in ( b , f ) n = 10 representative ROIs, in ( c , g ) n = 3 (independent experiments), one-way ANOVA.

Article Snippet: Human prostate cell PNT2 (ECACC 95012613), LNCaP (clone FCG, ECACC 89110211) and PC3 (ECACC 90112714) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) from CellBank Australia (Children's Medical Research Institute, Westmead, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Membrane, Staining

Journal: Journal of Neurochemistry

Article Title: A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis

doi: 10.1111/jnc.12029

Figure Lengend Snippet: Competition radioligand binding on tsA201 cells transiently expressing with human α7 nicotinic acetylcholine receptors (nAChRs). Equilibrium radioligand binding was performed with [ 3 H]-α-bungarotoxin (1 nM). Spinosad caused no significant displacement of [ 3 H]-α-bungarotoxin binding, whereas MLA caused complete displacement of specific radioligand binding ( K i = 4.2 ± 0.2 nM). Data points are means of triplicate samples (± SEM) from a single experiment, and data are typical of three independent experiments.

Article Snippet: Radioligand binding to transiently transfected tsA201 cells was performed essentially as described previously (Lansdell and Millar ).

Techniques: Binding Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3 and JPH4 interact with the Ca V 2.1 II-III loop. Mid-level confocal scans of tsA201 cells transfected with mCherry-tagged JPH3 or JPH4 together with the isolated, GFP-tagged Ca V 2.1 cytoplasmic domains: N-terminus ( A ), I-II loop ( B ), II-III loop ( C ), III-IV loop ( D ), and C-terminus ( E ). For each combination of constructs, the three panels arrayed vertically illustrate the distribution of the junctophilin ( red , topmost), cytoplasmic domain ( green , center), and the overlay of these two images ( bottom -most). At the cell periphery, the Ca V 2.1 II-III loop colocalized with both JPH3 and JPH4, as revealed in the overlaid images by the presence of yellow puncta, some of which are indicated by arrowheads ( C ). There appeared to be no colocalization of any other Ca V 2.1 cytoplasmic domains with either JPH3 or JPH4. Bars represent 5 μm. F , Pearson's colocalization coefficients calculated from confocal sections at the bottom surface of the cell. Data are reported as values from individual cells ( circles ), with the mean indicated by the height of the superimposed rectangles and ± SD by the horizontal black lines. For each construct combination, the numbers indicate total number of analyzed cells/number of separate transfected dishes. The colocalization between the II-III loop and JPH4 appeared to be somewhat greater than that between the loop and JPH3 (∗∗ p = 0.0019, t test with Welch's correction).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Transfection, Isolation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3 and JPH4 lacking the ER transmembrane segment retain the ability to interact with the Ca V 2.1 II-III loop, and this interaction appears to rescue junction formation. A , schematic representation of JPH-ΔTM constructs, JPH3 1-707 and JPH4 1-576 , both of which lack the TM domain that anchors them to the ER but retain the N-terminal regions responsible for association with the plasma membrane. B , bottom surface optical sections of tsA201 cells transfected with mCherry-JPH3 1-707 or mCherry-JPH4 1-576 alone ( a ), full-length mCherry-JPHs alone ( b ), and mCherry-JPH3 1-707 or mCherry-JPH4 1-576 in combination with the GFP-Ca V 2.1 II-III loop ( c ). In the absence of the II-III loop, the truncated junctophilins were distributed evenly on the plasma membrane ( a ). The interaction between the II-III loop, which is associated with the ER, and the JPH-ΔTMs, which are associated with the plasma membrane, was strong enough to rescue ER-PM junction formation, which caused redistribution of the truncated junctophilins into a punctate pattern ( c ) resembling the one observed for full-length junctophilins ( b ). C , mid-level confocal scans of tsA201 cells transfected with mCherry-JPH3 1-707 or mCherry-JPH4 1-576 in combination with the GFP-Ca V 2.1 II-III loop. Bars represent 5 μm in ( B ) and (C). D , Pearson's colocalization coefficients for the specified combinations of II-III loop and junctophilin constructs were calculated from optical sections of the bottom of the cell. Data are reported as values from individual cells ( circles ), with the mean indicated by the height of the superimposed rectangles, and ± SD by the horizontal black lines ( p = 0.0613, t test with Welch's correction). Numbers in parentheses represent total number of analyzed cells/separate transfected dishes.

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Construct, Clinical Proteomics, Membrane, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3-ΔTM and JPH4-ΔTM both interact with the synprint-containing, N-terminal half of the Ca V 2.1 II-III loop. Only JPH4-ΔTM interacts with the C-terminal half of the II-III loop. A , top : schematic representation of Ca V 2.1 II-III 715-1084 with the synprint domain indicated in red . Bottom : midlevel image of a tsA201 cell transfected only with GFP-tagged II-III 715-1084 . B , midlevel optical sections of tsA201 cells expressing mCherry-JPH3 1-707 or mCherry-JPH4 1-576 together with GFP-Ca V 2.1 II-III 715-1084 . Co-expression with either JPH3 1-707 or JPH4 1-576 caused Ca V 2.1 II-III 715-1084 to become concentrated at the cell periphery, where it colocalized with the JPH-ΔTM constructs. C , Pearson's coefficients calculated from midlevel optical scans. (n.s. p = 0.478, t test with Welch's correction). D , top : schematic representation of Ca V 2.1 II-III 1037-1253 . Bottom : midlevel image of a tsA201 cell transfected only with GFP-tagged II-III 1037-1253 . E , midlevel optical sections of tsA201 cells expressing mCherry-JPH3 1-707 or mCherry-JPH4 1-576 together with GFP-II-III 1037-1253 . Ca V 2.1 II-III 715-1084 did not colocalize with JPH3 1-707 but did colocalize with JPH4 1-576 . F , Pearson's coefficients calculated from bottom surface scans (∗∗∗∗ p < 0.0001, t test with Welch's correction). In ( C ) and ( F ), circles indicate values for individual cells, with the mean ± SD indicated by the superimposed rectangle and horizontal black lines, respectively; numbers in parentheses indicate the total number of analyzed cells/number of separate transfected dishes. Bars represents 5 μm in ( A ), ( B ), ( D ), and ( E ).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Transfection, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: Recruitment to ER-PM junctions and slowing of inactivation of the Ca V 2.1 Δ2-like variant, which lacks most of the synprint domain, are mediated by JPH4 but not by JPH3. A , schematic representation of rabbit Ca V 2.1 showing the synprint domain ( red ), with its first and last residues indicated. The cyan bracket and numbers indicate the residues deleted to obtain the Δ2-like rabbit variant, based on the rat Δ2 splice variant and the alignment illustrated in Fig. S3 . B , midlevel optical sections of tsA201 cells transfected with GFP-Ca V 2.1 Δ2-like variant, β1b, α2δ1 in combination with either mCherry-JPH3 ( left column) or mCherry-JPH4 ( right column). Accumulation of the channel at JPH4-induced junction is visible ( white arrowheads). C , Pearson's colocalization coefficients for the specified combinations of Ca V 2.1 and junctophilins, calculated from optical sections of the bottom of the cell. Pearson’s coefficients for Ca V 2.1 with the intact synprint domain are part of a data set previously described in Perni and Beam, 2021 . All data are reported as values from individual cells ( circles ), with the mean indicated by the height of the superimposed rectangles, ± SD [2-way ANOVA: “no JPH v.s. JPH3”: p = 0.93, F (1,110) = 0.008. 2-way ANOVA “no JPH” v.s. “JPH4”, p < 0.0001, F (1, 111) = 172.9; post hoc Sidak's test: ∗∗ p = 0.0035, n.s. p = 0.135]. D , percentage of peak Ca 2+ current remaining 700 ms after the peak (I 700 /I peak ) is plotted (mean ± SD) as a function of test potential in tsA201 cells transfected with GFP-tagged Ca V 2.1 Δ2-like variant, β1b and α2δ1 together with either no junctophilins ( black ), JPH3 ( blue ), or JPH4 ( gold ), which were tagged with mCherry. The inset illustrates representative Ca 2+ currents (scaled to match in amplitude) elicited by an 800 ms depolarization to +50 mV, with the vertical dotted line indicating the current 700 ms after the peak. On average, the inactivation of the Ca V 2.1 Δ2-like variant was unaffected by JPH3 but significantly reduced by JPH4 (2-way ANOVA, post hoc Sidak's test: ∗ p = 0.032; ∗∗ p = 0.002; ∗∗∗∗ p < 0.0001). Numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. Bars represent 2 μm.

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Variant Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3 and JPH4 constructs lacking the divergent and TM domains interact with the Ca V 2.1 II-III loop, but this interaction is lost after the additional removal of all or most of the α-helical domain. A , schematic representation of C-terminally truncated JPH constructs JPH3 1-423 and JPH4 1-417 , in which the divergent and transmembrane domains have been removed and midlevel optical sections of tsA201 cells expressing either of these two mCherry-tagged constructs in combination with GFP-tagged Ca V 2.1 II-III loop. The II-III loop colocalized with both JPH3 1-707 and JPH4 1-576, as indicated by the yellow segments at the cell periphery in the overlaid images ( e.g. , yellow arrowheads). Pearson's coefficients for these construct combinations, calculated from bottom -surface scans, are plotted in ( B ). C , schematic representation of JPH3 1-336 and JPH4 1-364 in which C-terminal truncation removed all, or part, respectively, of the α-helical domain and midlevel optical sections of tsA201 cells expressing mCherry-JPH3 1-336 or mCherry-JPH4 1-364 together with the GFP-tagged Ca V 2.1 II-III loop. There was no apparent colocalization of the II-III loop with either of these constructs. Pearson's coefficients, calculated from bottom-surface scans, are plotted in ( D ). In ( B ) and ( D ), circles indicate values for individual cells, with the mean ± SD indicated by the superimposed rectangle and horizontal black lines, respectively. Numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. ∗∗∗∗ p < 0.0001; n.s. p = 0.84 ( t test with Welch's correction). Bars represent 5 μm in ( A ) and ( C ).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Construct, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: The α-helical domain is responsible for the ability of JPH4 to interact with the distal half of the Ca V 2.1 II-III loop. A , schematic representation of constructs JPH3 1-333 -JPH4 α-Hlx , which contains the MORN and joining domains of JPH3 fused with the α-helical domain of JPH4, and the complementary construct JPH4 1-327 -JPH3 α-Hlx , containing the MORN and joining domains of JPH4 and the α-helical domain of JPH3. The first and last residues of the swapped α-helical domains are indicated in gold (JPH4 α-helical domain) and blue (JPH3 α-helical domain). B , midlevel optical sections of tsA201 cells expressing mCherry-tagged JPH3 1-333 -JPH4 α-Hlx or JPH4 1-327 -JPH3 α-Hlx together with the full-length Ca V 2.1 II-III loop tagged with GFP. The full-length II-III loop colocalized at the surface with both these junctophilin chimeras ( yellow segments in overlays, some indicated by arrowheads). Pearson's coefficients for these construct combinations, calculated from bottom-surface scans, are plotted in ( C ). D , midlevel optical sections of tsA201 cells expressing mCherry-tagged JPH3 1-333 -JPH4 α-Hlx or JPH4 1-327 -JPH3 α-Hlx together with the distal half of the Ca V 2.1 II-III loop, II-III 1037-1253 , tagged with GFP. Unlike the full-length II-III loop, which showed clear colocalization with both junctophilin chimeras, the only apparent colocalization of the distal half of the II-III loop was with JPH3 1-333 -JPH4 α-Hlx . Pearson's coefficients for these combinations of constructs, calculated from bottom-surface scans, are plotted in ( E ). In ( C ) and ( E ), circles indicate values calculated for individual cells, with the mean ± SD indicated by the superimposed rectangle and horizontal black lines, respectively. Numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. In ( C ) and ( E ), n.s p = 0.548; ∗∗∗∗ p < 0.0001 ( t test with Welch's correction). Bars represent 5 μm in ( B ) and ( D ).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Construct, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: The greater slowing of Ca V 2.1 inactivation by JPH4 than by JPH3 is not attributable to the α-helical domain. A , representative Ca 2+ currents (scaled to match in amplitude) elicited by an 800 ms depolarization to +40 mV in tsA201 cells transfected with YFP-Ca V 2.1, β1b, and α2δ1 together with either no junctophilins ( black ) or with mCherry-tagged JPH3 ( blue ), JPH4 ( gold ), JPH3 1-333 -JPH4 α-Hlx ( orange ), or JPH4 1-327 -JPH3 α-Hlx ( green ) designated in the figure as JPH3-JPH4 α-Hlx and JPH4-JPH3 α-Hlx , respectively. The vertical dotted line indicates the current 700 ms after the peak. B , percentage of peak current remaining 700 ms after the peak (I 700 /I peak ) is plotted (mean ± SD) as a function of test potential for Ca 2+ currents recorded from tsA201 cells transfected with Ca V 2.1, β1b, and α2δ1 together with either no junctophilins ( black ), JPH3 1-333 -JPH4 α-Hlx ( orange ), or JPH4 1-327 -JPH3 α-Hlx ( green ). In ( B ), numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. The currents for JPH3 and JPH4, and the current and I 700 /I peak values for no junctophilins, are part of a data set previously described in Perni and Beam, 2021 [2-way ANOVA: “JPH4 1-327 -JPH3α-Hlx v.s. JPH3 1-333 -JPH4α-Hlx”: p < 0.0001, F (1,168) = 197.9; post hoc Sidak's test: ∗∗∗ p = 0.0004; ∗∗∗∗ p < 0.0001].

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Transfection

Journal: Cell Reports

Article Title: The α 2 δ-like Protein Cachd1 Increases N-type Calcium Currents and Cell Surface Expression and Competes with α 2 δ-1

doi: 10.1016/j.celrep.2018.10.033

Figure Lengend Snippet:

Article Snippet: For co-IPs and electrophysiological studies, tsA-201 cells were transfected using Fugene6 (Promega, Fitchburg, WI) according to the manufacturer’s protocol.

Techniques: Affinity Purification, Recombinant, Modification, Nucleic Acid Electrophoresis, Membrane, Plasmid Preparation, Bradford Assay, Mutagenesis, Software