Journal: eLife

Article Title: GPC1 specific CAR-T cells eradicate established solid tumor without adverse effects and synergize with anti-PD-1 Ab

doi: 10.7554/eLife.49392

Figure Lengend Snippet:

Article Snippet: Transfected construct (Gallus gallus domesticus , Homo-sapiens s ) , hCAR-HL , This paper, see Materials and methods and , , .

Techniques: Derivative Assay, Expressing, Plasmid Preparation, Transfection, Construct, Activation Assay, In Vivo, Injection, Enzyme-linked Immunosorbent Assay, Sequencing, Recombinant, Software

Journal: eLife

Article Title: Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9

doi: 10.7554/eLife.54100

Figure Lengend Snippet:

Article Snippet: Transfected construct (human) , Opossum TLR4 , Genscript , (UniProt #F6Y6W8) , In pcDNA3.1(+) backbone.

Techniques: Software, Sequencing, Recombinant, Plasmid Preparation, Expressing, Mutagenesis, Transfection, Construct, Luciferase

Journal: eLife

Article Title: Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

doi: 10.7554/eLife.63614

Figure Lengend Snippet: ( A ) Vectors encoding the indicated inner nuclear membrane (INM) proteins were transfected into HeLa cells. Cells were fixed after 24 or 48 hr and analyzed by confocal microscopy. Scale bar, 10 μm. ( B ) Time-lapse images of LAP1B-GFP or LAP2β-GFP expressing HeLa cells progressing through mitosis. Expression of the constructs was induced 24 hr prior to imaging. DNA was visualized by SirHoechst and used to define anaphase onset (t = 00:00 hr:min). Scale bar, 5 μm. ( C ) Scheme depicting the two human LAP1 isoforms, LAP1B and LAP1C. ( D ) Representative images of stable HeLa cells lines expressing LAP1B-GFP and LAP1C-GFP after induction with different tetracycline (tet) concentrations for 48 hr. Please note that we observed some ‘twin’ nuclei upon overexpression of LAP1B (white arrow), which can be a sign of binucleation, as further analyzed in . Scale bar, 10 μm. ( E ) LAP1 levels at the NE of cells from the experiment shown in panel D were analyzed by quantification of the integrated GFP density (IntDen GFP) per nucleus. ( F ) LAP1 levels in cell lysates derived from the experiment shown in panel D were analyzed by immunoblotting. Note that the LAP1B-GFP cell line expressed LAP1C-GFP independent of tetracycline addition, due to an alternative transcriptional start site used for the production of the shorter LAP1 isoform . ( G ) Left: Maximum intensity z-projections (5 × 0.63 μm) of confocal images from fixed metaphase HeLa cells expressing LAP1B-GFP or LAP1C-GFP after 48 hr of induction. Scale bar, 5 μm. Right: Confocal images of fixed HeLa cells in interphase. Scale bar, 10 μm.

Article Snippet: Transfected construct (human) , si-Tor1B , Qiagen/Microsynth , , 5’- CTGTCGGAGTCTTCAATAATA-3’.

Techniques: Transfection, Confocal Microscopy, Expressing, Construct, Imaging, Over Expression, Derivative Assay, Western Blot

Journal: eLife

Article Title: Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

doi: 10.7554/eLife.63614

Figure Lengend Snippet: ( A ) Vector encoding LAP1B-GFP was transiently transfected into HCT116 or HepG2 cells. Cells were fixed after 48 hr and analyzed by confocal microscopy. Scale bars, 10 μm. ( B ) Western blot analysis (left) and integrated GFP fluorescence intensities (right) in LAP1B-GFP and LAP2β-GFP-expressing cells of the experiment depicted in . ( C ) Comparison of the metaphase localization of nuclear lamins and select inner nuclear membrane (INM) proteins in an inducible LAP1B-GFP cell line without and with tetracycline (tet) induction. Maximum intensity projections (5 × 0.63 μm). Scale bar, 5 μm.

Article Snippet: Transfected construct (human) , si-Tor1B , Qiagen/Microsynth , , 5’- CTGTCGGAGTCTTCAATAATA-3’.

Techniques: Plasmid Preparation, Transfection, Confocal Microscopy, Western Blot, Fluorescence, Expressing

Journal: eLife

Article Title: Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

doi: 10.7554/eLife.63614

Figure Lengend Snippet: ( A ) HeLa cells were transiently transfected with constructs encoding for LAP1B-GFP or LAP1B(1-359), either alone or together with the depicted Torsin constructs. After 48 hr, cells were fixed, subjected to immunostaining of Torsins using an anti-HA antibody and analyzed by confocal microscopy. Scale bar, 10 μm. ( B ) Representative images of pre-defined classes of cell nuclei used for the classification of NE aberration phenotypes. Based on nuclear shape, cells were assigned into one of the five shown categories for the quantification in C. The two upper classes were regarded as normal (no; no NE aberration), and the three lower classes as aberrant (yes). Scale bar, 5 μm. ( C ) The percentage of cells with NE aberrations was quantified by assigning cells into one of five predefined phenotypic classes shown in B. (left; N = 3, n > 103, mean +/- SEM). Only cells with similar LAP1B-GFP expression levels, based on quantification of the integrated GFP density per cell (IntDen GFP, normalized to the LAP1B minus Torsin control; right) were considered. ( D ) Depiction of the mutant LAP1B constructs; R563G: Torsin activation deficient-mutant; E482A, disease-causing mutant. ( E ) HeLa cells were transiently transfected with constructs encoding for the indicated LAP1B-GFP derivatives, either alone or together with Tor1B-mRFP. After 48 hr, cells were fixed and analyzed by confocal microscopy. Scale bar, 10 μm. ( F ) The fraction of cells with NE aberrations was quantified as in C (N = 3, n > 114, mean +/- SEM).

Article Snippet: Transfected construct (human) , si-Tor1B , Qiagen/Microsynth , , 5’- CTGTCGGAGTCTTCAATAATA-3’.

Techniques: Transfection, Construct, Immunostaining, Confocal Microscopy, Expressing, Mutagenesis, Activation Assay

Journal: eLife

Article Title: Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

doi: 10.7554/eLife.63614

Figure Lengend Snippet: ( A ) HeLa cells were transfected with constructs encoding for LAP1B-GFP, either alone or together with LULL1-HASt and Tor1B-mRFP expression vectors. Cell were fixed 48 hr after transfection and nuclear morphology analyzed by confocal microscopy. Scale bar, 10 μm. ( B ) Quantification of NE aberrations in cells with similar LAP1B-GFP expression levels as in (N = 3, n > 99, mean +/- SEM).

Article Snippet: Transfected construct (human) , si-Tor1B , Qiagen/Microsynth , , 5’- CTGTCGGAGTCTTCAATAATA-3’.

Techniques: Transfection, Construct, Expressing, Confocal Microscopy

Journal: eLife

Article Title: Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

doi: 10.7554/eLife.63614

Figure Lengend Snippet:

Article Snippet: Transfected construct (human) , si-Tor1B , Qiagen/Microsynth , , 5’- CTGTCGGAGTCTTCAATAATA-3’.

Techniques: Transfection, Construct, Recombinant, Molecular Cloning, Knock-Out

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: ( A ) Schematic representation of JPH3 and JPH4, indicating the ‘MORN’ motifs that bind to the plasma membrane and C-terminal segment (‘TM’) that traverses the ER membrane. Except for the ‘Divergent’ domain, the two proteins display substantial sequence similarity . ( B ) Confocal optical sections acquired at mid-level (top row) or the bottom surface (middle row), and thin section electron micrographs (bottom row), are shown for cells transfected with mCherry-JPH3 or mCherry-JPH4 (left and right columns, respectively). The fluorescence was predominantly present in discrete foci near the cell periphery as expected for ER-PM junctions, which can be directly visualized in the electron micrographs in which the junctional gap and the ER sub-cortical cisternae are pseudo-colored in purple and yellow, respectively. Scale bars = 5 μm (top row), 2 μm (middle row), and 100 nm (bottom row).

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Clinical Proteomics, Membrane, Sequencing, Transfection, Fluorescence

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Mid-level or bottom-surface optical sections are shown for tsA201 cells transfected with the designated Ca V constructs (represented in green) either in the absence of junctophilins ( A ) or together with either mCherry-JPH3 ( B ) or mCherry-JPH4 ( C ), in which the junctophilins are represented in red in merged red/green images. cDNAs for the auxiliary subunits β1b and α2-δ1 were also present for the high-voltage-activated channels. For the three high-voltage-activated channels, co-expression with JPH3 or JPH4 resulted in clusters of channels that were near the surface and colocalized with the junctophilins. Conversely, Ca V 3.1 localization was unaffected by the presence of the two JPHs. Scale bars = 5 and 2 μm, respectively, for the mid-level and bottom-surface images. ( D–G ) Pearson's colocalization coefficients for the specified combinations of neuronal calcium channels and junctophilins, which were calculated from optical sections of the bottom of the cell that was adjacent to the substrate (see B and C for examples). In this plot (and subsequent plots), individual data points indicate Pearson's coefficient for a single cell, with the mean and ± SEM for each construct combination indicated by longer and shorter horizontal lines, respectively. Numbers of cells are indicated in parentheses. Pearson’s coefficients and their statistical comparison are provided in . Figure 2—source data 1. Numerical data and statistical analyses to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Transfection, Construct, Expressing, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Ca 2+ currents were measured in tsA201 cells transfected with Ca V 1.2 ( A ), Ca V 2.1 ( B ), or Ca V 2.2 ( C ) either without junctophilins (black) or together with either JPH3 (red) or JPH4 (green). Constructs for β1b and α2-δ1 were also present. The upper row of panels illustrates the average peak current versus voltage relationships and the lower row of panels plots the percentage of peak current remaining 700 ms after the peak (I 700 /I peak ) as a function of test potential. The insets illustrate representative currents (scaled to match in height) elicited by an 800 ms depolarization to the indicated potential, with the current 700 ms after the peak indicated by the vertical dotted line. Data are shown as mean ± SEM. Tables of I peak and I 700 are provided in . Figure 3—source data 1. Numerical data to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Transfection, Construct

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Ba 2+ currents were measured in tsA201 cells transfected with Ca V 1.2 ( A ), Ca V 2.1 ( B ), or Ca V 2.2 ( C ) either without junctophilins (open symbols) or together with JPH4 (closed symbols). Constructs for β1b and α2-δ1 were also present. The percentage of peak current remaining 700 ms after the peak (I 700 /I peak ) is plotted as a function of test potential. Data are shown as mean ± SEM. Tables of I peak and I 700 are provided in . Figure 3—figure supplement 1—source data 1. Numerical data to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Transfection, Construct

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Cells were transfected with the calcium channels together with JPH3(1 – 707) or JPH4(1 – 576), which lack the ER-spanning membrane segment and thus associate with the cell surface without inducing ER-PM junctions. ( A, B ) Peak current and I 700 /I peak as a function of test potential for Ca V 2.1 and Ca V 2.2, respectively, expressed without junctophilin, with JPH3(1 – 707) or JPH4(1 – 576), indicated in black, purple, and blue, respectively. cDNAs for the Ca V auxiliary subunits β1b and α2-δ1 were also present. The insets illustrate representative Ca 2+ currents, which were elicited by 800 ms depolarizations to +30 mV and scaled to match in peak height. On average, the truncated junctophilins slowed inactivation to an extent that was comparable to, or greater than, that caused by the full-length junctophilins . Data are shown as mean ± SEM. Tables of I peak and I 700 are provided in . Figure 4—source data 1. Numerical data to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Transfection, Membrane

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Representative, red/green merged images of the mid-level or bottom surface of tsA201 cells expressing GFP-RyR1, YFP-RyR2, or YFP-RyR3 (left to right, represented in green) together with either mCherry-JPH3 ( A ) or mCherry-JPH4 ( B ), which are represented in red. Scale bars = 5 and 2 μm, respectively, for the mid-level and bottom-surface images. ( C ) Pearson's colocalization coefficients for the specified combinations of junctophilins and RyRs, which were calculated from bottom-surface optical sections. Statistical significance: ****p < 0.0001, *p = 0.0248, p = 0.2366 (ns). Pearson’s coefficients and their statistical comparison are provided in . Figure 5—source data 1. Numerical data and statistical analyses to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Expressing, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Pearson’s coefficients, calculated from bottom-surface scans, for mCherry-JPH3 versus YFP-RyR2 (left), and for mCherry-JPH4 versus YFP-RyR3 (right) expressed either alone or together with CFP-Ca V 1.2 plus β1b and α2-δ1. Statistical significance: p > 0.4 (ns). Pearson’s coefficients and their statistical comparison are provided in . Figure 6—source data 1. Numerical data and statistical analyses to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Ca 2+ release from the endoplasmic reticulum (ER) is detectable after depolarization of cells expressing RyR1, JPH3, and either Ca V 2.1 or Ca V 2.2. ( A ) Representative images of cells expressing YFP-Ca V 2.1 (represented in red), CFP-RyR1 (represented in green), and either mCherry-JPH3 or mCherry-JPH4 (represented in cyan) as indicated. The rightmost images in each row are overlays of the YFP-Ca V 2.1 and CFP-RyR1 images. Pearson’s coefficients for these combinations of constructs calculated from bottom-surface scans are plotted in ( B ). ( C ) Representative images of cells expressing YFP-Ca V 2.2 (represented in red), CFP-RyR1 (represented in green), and either mCherry-JPH3 or mCherry-JPH4 (represented in cyan), as indicated. The rightmost images in each row are overlays of the YFP-Ca V 2.2 and CFP-RyR1 images. Pearson’s coefficients for these combinations of constructs, calculated from bottom-surface scans, are plotted in ( D ). In all cases ( A–D ), the cells were also transfected with β1b and α2-δ1. Statistical significance for ( B ) and ( D ): ****p ≤ 0.0001. ( E–J ) Cells stably transfected with RyR1 were transiently transfected with CFP-JPH3, R-CEPIAer (represented in red), β1b, α2-δ1, and either Ca V 2.1 ( E–G ) or Ca V 2.2 ( H–J ) and loaded with Fluo8-AM (represented in green). Representative images of such cells, acquired prior to depolarization, are shown in ( E ) and ( H ) for Ca V 2.1 and Ca V 2.2, respectively. ( F, I ) The Fluo8 and R-CEPIAer fluorescence for these two cells within the indicated regions of interest (outlined in yellow) is plotted as a function of time in response to a 2.5 s, focal application of 100 mM KCl. 4 of the 12 cells producing Fluo8 transients for Ca V 2.1 also displayed decreased ER Ca 2+ . 4 of the 11 cells producing Fluo8 transients for Ca V 2.2 also displayed decreased ER Ca 2+ . ( G, J ) Average responses of the four ER-responding cells, for each of the two construct combinations, represented as mean (solid lines) ± SEM (thin vertical lines). Pearson’s coefficients plotted in ( B ) and ( D ) and their statistical comparison are provided in . Raw data for ΔF/F 0 plotted in ( F ), ( G ), ( I ), and ( J ) are given in . Figure 7—source data 1. Numerical data and statistical analyses to support graphs in . Figure 7—source data 2. Numerical data to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Expressing, Construct, Transfection, Stable Transfection, Fluorescence, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: ( A ) Schematic representation of the chimera ‘JPH3-with-JPH4-divergent’ in which the divergent domain of JPH3 has been replaced by that from JPH4. ( B ) Red only (top row) and red/green merged images (bottom row) of mid-level confocal sections of tsA201 cells expressing the mCherry-tagged chimera, illustrated in ( A ), together with GFP-RyR1, YFP-RyR2, or YFP-RyR3 (left to right, represented in green). Scale bar = 5 μm. ( C ) Pearson's coefficients for colocalization between the three RyR isoforms and the chimeric junctophilin (center) calculated from bottom-surface images, compared with those for JPH3 (left) and JPH4 (right), which are replotted from . Statistical significance: ***p ≤ 0.001, p > 0.99 (ns). Pearson’s coefficients are listed in , together with their statistical comparison to one another and to Pearson’s coefficients plotted in . Figure 8—source data 1. Numerical data and statistical analyses to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Expressing, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: ( A ) Pearson's coefficients for tsA201 cells co-transfected with YFP-Ca V 1.2 and mCherry-tagged JPH3, JPH3-with-JPH4-divergent (see ), or JPH4. Constructs for β1a and α2δ1were also present. ( B ) Fractional decay of calcium current (I 700 /I peak ) in cells transfected with YFP-Ca V 2.1 or GFP-Ca V 2.2 either without junctophilins or together with the indicated mCherry-tagged junctophilin constructs. cDNAs for the Ca V auxiliary subunits β1b and α2-δ1 were also present. JPH3-with-JPH4-divergent is very similar to intact JPH3 both in recruitment of Ca V 1.2 and in the slowing of inactivation of Ca V 2.1 and Ca V 2.2. Pearson’s coefficients plotted in ( A ) and their statistical comparison are listed in . I peak and I 700 plotted in ( B ) are given in . Figure 8—figure supplement 1—source data 1. Numerical data and statistical analyses to support graph in . Figure 8—figure supplement 1—source data 2. Numerical data to support graph in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Transfection, Construct, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: Mid-level and bottom-surface optical sections of tsA201 cells expressing YFP-RyR1 1:4300 , YFP-RyR2 1:4226, or YFP-RyR3 1:4032 (represented in green) together with either mCherry-JPH3 ( A ) or mCherry-JPH4 ( B ). The mid-level sections are illustrated both as overlaid red/green images and green-only images (first and second rows, respectively). Note that in the presence of JPH4 YFP-RyR1 1:4300 , YFP-RyR2 1:4226 , and YFP-RyR3 1:4032 all behaved as large cytoplasmic proteins, which were excluded from the nucleus and lumen of the ER but were otherwise uniformly distributed. In cells co-expressing JPH3 and RyR3 1:4032 , some junctions contained both proteins, whereas others contained JPH3 with little RyR3 1:4032 (indicated by arrowheads in the lower-right panel of A ). Scale bars = 5 and 2 μm, respectively, for the mid-level and bottom-surface images. ( C ) Schematic representation of an RyR monomer, indicating the approximate position at which the large cytoplasmic domain was severed from the ER-traversing segments identified in the cryo-EM structures ( ; ). ( D–F ) Pearson's colocalization coefficients for the specified construct combinations calculated from bottom-surface images. Statistical significance: ****p < 0.0001, p > 0.99 (ns). Pearson’s coefficients and their statistical comparison are provided in . Figure 9—source data 1. Numerical data and statistical analyses to support graphs in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Expressing, Cryo-EM Sample Prep, Construct, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet: ( A–E ) Confocal sections of tsA201 cells transfected with YFP-RyR1 1:4300 (represented in green) and the indicated mCherry-tagged constructs. For all the constructs, the leftmost image displays only the mCherry fluorescence (in red), and the image just to its right is a red/green overlay of the mCherry and YFP fluorescence. ( A, B ) The constructs JPH3(1 – 707) and JPH4(1 – 414)-JPH3(418 – 707) lack the endoplasmic reticulum (ER) transmembrane domain but have MORN motifs that cause association with the plasma membrane (leftmost images). The distribution of YFP-RyR1 1:4300 was similar for JPH3(1 – 707) and JPH4(1 – 414)-JPH3(418 – 707): it overlapped the junctophilin constructs at the cell surface and was also diffusely present in the cytoplasm (second images from left). After photobleaching the YFP tag within the area outlined in violet, YFP-RyR1 1:4300 remained concentrated at the cell surface as indicated both in the red/green overlays (third images from left) and in the images of only the YFP-RyR1 1:4300 fluorescence (rightmost images). See text for additional details. ( C, D ) mCh-JPH3(418 – 748) and JPH3(653 – 748)-mCh lack the MORN motifs required for association with the plasma membrane and were distributed in a reticular pattern in the cell interior (left), with the YFP-RyR1 1:4300 having an overlapping pattern (right). ( E ) YFP-RyR1 1:4300 did not colocalize with mCherry-ER. Scale bars = 5 μm. ( F ) Pearson colocalization coefficients for YFP-RyR1 1:4300 versus the indicated constructs, calculated from mid-level optical sections. In the case of JPH3(1 – 707) and JPH4(1 – 414)-JPH3(418 – 707), these were calculated from sections acquired after photobleaching of YFP-RyR1 1:4300 in the cell interior. **** Significantly smaller than RyR1 1:4300 versus the other four junctophilin constructs. Pearson’s coefficients and their statistical comparison are provided in . Figure 10—source data 1. Numerical data and statistical analyses to support graph in .

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Transfection, Construct, Fluorescence, Clinical Proteomics, Membrane, Comparison

Journal: eLife

Article Title: Neuronal junctophilins recruit specific Ca V and RyR isoforms to ER-PM junctions and functionally alter Ca V 2.1 and Ca V 2.2

doi: 10.7554/eLife.64249

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo sapiens ) , JPH4 , GenScript , C97908 , In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS.

Techniques: Stable Transfection, Expressing, Transfection, Construct, Plasmid Preparation, Modification, Recombinant, Sequencing, Software