Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Impact of indicated factors/features (e.g. protein-protein interactions [PPIs], post-translational modifications [PTM], cis-regulatory elements [CRE]) on the switch-like behavior of kinases. ( B ) Schematic representation of the KinCon reporter technology using the Renilla luciferase ( R Luc) protein-fragment complementation assay (PCA) as it applies to kinases such as BRAF which contain auto-inhibitory modules (AIM); R Luc fragments 1 and 2 are N and C terminally fused to the kinase of interest (with interjacent linker in red) and are labeled with F[1] and F[2]. PPIs, drug (candidate) or small molecule binding, mutations and/or PTMs may convert the KinCon reporter into different conformation states. Protein movements are quantified through measuring alterations of bioluminescence signals upon R Luc substrate addition. ( C ) Shown is the workflow for the KinCon reporter construct engineering and analyses using KinCon technology. The kinase gene of interest is inserted into the multiple cloning site of a mammalian expression vector which is flanked by respective PCA fragments (F[1]-, -F[2]; KD, kinase domain) and separated with interjacent flexible linkers. Expression of the genetically encoded reporter in indicated multi-well formats allows to vary expression levels and define a coherent drug treatment plan. Moreover, it is possible to alter the kinase sequence (mutations) or to co-express or knock down the respective endogenous kinase, interlinked kinases, or proteinogenic regulators of the respective pathway. After systematic administration of pathway modulating drugs or drug candidates, analyses of KinCon structure dynamics may reveal alterations in potency, efficacy, and potential synergistic effects of the tested bioactive small molecules (schematic dose-response curves are depicted). ( D ) Simplfied schematic representation of the activation mechanisms of BRAF, LKB1, RIPK1, and CDK6 complexes (with indication of selected regulators or complex components) engaged in altering OFF (top) or ON (bottom) kinase states. ( E ) Representative KinCon experiments of time-dependent expressions of indicated KinCon reporter constructs in HEK293T cells are shown (mean ± SEM). Indicated KinCon reporters were transiently over-expressed in 24-well format in HEK293T cells for 10 hr, 16 hr, 24 hr, and 48 hr each. Immunoblotting show expression levels of endogenous kinases and over-expressed KinCon reporters. ( F ) Impact of 1 μM PLX8394 exposure (for 1 hr) on BRAF and BRAF-V600E KinCon reporters (HEK293T cells) is shown. Representative experiment of n=4 independent is presented. ( G ) R Luc PCA values have been normalized on the untreated conditions. The mean ± SEM of PLX8394 exposure on BRAF conformation opening and closing of n=4 experiments is shown. RLU, relative light units. Statistical significance for G: one-sample t-test (*p<0.05, **p<0.01, ***p<0.001). Figure 1—source data 1. Raw unedited western blots shown in . Figure 1—source data 2. Uncropped and labelled western blots shown in .

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Luciferase, Protein-Fragment Complementation Assay, Labeling, Binding Assay, Construct, Clone Assay, Expressing, Plasmid Preparation, Sequencing, Knockdown, Activation Assay, Western Blot

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Summary of n=4 independent experiments of time-dependent expressions of indicated KinCon reporter constructs in HEK293T cells is shown (mean ± SEM). BRAF-V600E, LKB1, RIPK1, and CDK6 KinCon reporters were transiently over-expressed in 24-well format in HEK293T cells for 10 hr, 16 hr, 24 hr, and 48 hr each. ( B ) Impact of 1 μM PLX8394 exposure for 1 hr on BRAF and BRAF-V600E KinCon reporters transiently over-expressed for indicated time frames in HEK293T cells is shown. Mean of n=4 independent experiments with ± SEM is presented.

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Construct

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Simplified representation of the LKB1-complex composition which promotes AMPKα signaling via phosphorylation at position Thr172. ( B ) Crystal structure of the LKB1-STRADα-MO25 complex (PDB code 2WTK, ) representing a snapshot of trimeric complex assembly. The missense mutations we have analyzed are indicated in blue (STRADα) and pale yellow and rose (LKB1). The ATP analogue AMP-PNP is depicted in light green sticks. ( C ) Domain organization of human LKB1, STRADα, and MO25 (accession numbers: Q15831, Q7RTN6, Q9Y376) with indication of the kinase and pseudokinase domains (KD). Shown in red are tested missense mutations. These are summarized in the table together with their origin and assumed functions ( , , , , , , ). ( D ) Effect of co-expressions of indicated kinase complex components on AMPK phosphorylation (HeLa cells, 48 hr post transfection) (mean ± SEM, n=4 ind. experiments; 3x-Flag is indicated as flag). ( E ) Illustration of the kinase conformation (KinCon) reporter setup for STRADα KinCon measurements: Effect of LKB1-STRADα-MO25 complex formation on the STRADα KinCon reporter opening and closing (HEK293T cells, 48 hr post transfection). Expression corrected signals (STRADα-KinCon) are shown (mean ± SEM, n=4 ind. experiments). ( F ) KinCon reporter setup for LKB1 KinCon measurements: Effect of LKB1-STRADα-MO25 complex formation on the LKB1 KinCon reporter conformation. Expression corrected signals are shown (LKB1-KinCon; HEK293T cells, 48 hr post transfection) (mean ± SEM, n=5 ind. experiments). ( G ) LKB1-KinCon measurements upon co-expression of indicated proteins displaying the binding deficient STRADα mutations H231A/F233A (HF; see binding interface in B and H). Expression corrected signals are displayed (HEK293T cells, 48 hr post transfection) (mean ± SEM, n=4 ind. experiments). ( H ) Structure depiction highlights the localization of mutations conferring altered LKB1 functions. LKB1 residues K78, D176, and D194 (pale yellow sticks) are located within the catalytic cleft and in close proximity to AMP-PNP (light green sticks). ( I ) Impact of LKB1 missense mutations (three patient mutations D176N, D194N, and W308C and three ‘non-patient’ mutations K48R, R74A, K78I) on KinCon conformational changes upon co-expression of interactors. Expression corrected signals are displayed (HEK293T cells, 48 hr post transfection) (mean ± SEM, n=4 ind. experiments). Statistical significance for D, E, F, G, and I: one-sample t-test (*p<0.05, **p<0.01, ***p<0.001). Figure 2—source data 1. Raw unedited western blots shown in . Figure 2—source data 2. Uncropped and labelled western blots shown in .

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Transfection, Expressing, Binding Assay, Western Blot

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Simplified schematic representations of the activation pathways for apoptosis and necroptosis. Highlighted in black is the combination treatment termed TBZ (10 pg/ml TNFα, 10 nM BV-6, and 20 nM zVAD-FMK) that induces necroptosis. ( B ) Domain organization of human RIPK1 (accession number: Q13546), RIPK2 (accession number: O43353), RIPK3 (accession number: Q9Y572), and MLKL (accession number: Q8NB16). ( C ) Basal signals of indicated kinase conformation (KinCon) reporters following transient over-expression in HEK293T cells. Bars represent the RLU fold change relative to RIPK1 (mean ± SD, n=5 ind. experiments). ( D ) Time-dependent treatments using TBZ of HEK293T cells transiently expressing wild-type (wt) RIPK1 (left) and wt RIPK3 (right) KinCon reporters (expression corrected) (mean ± SD, n=3 ind. experiments). ( E ) Domain organization of RIPK1 displaying missense mutation sites. ( F ) 3D structure of RIPK1 dimers with functional mutations highlighted (PDB code: 6HHO, ). GSK547 is depicted as brown sticks. ( G ) KinCon reporter signals with/without mutations (S14/15/166A, S14/15/166E, K45A) were measured in a HEK293T RIPK1 knock-out (KO) cell line (expression corrected) (mean ± SEM, n=5 ind. experiments). ( H ) KinCon reporter signals of RIPK1 (patient loci: D324A, D324E, D324H, C601Y) were measured in HEK293T RIPK1 KO cells (expression corrected) (mean ± SD, n=5 ind. experiments). ( I ) 3D structure of RIPK1 with the inhibitor GSK547, which binds to an allosteric site in close proximity to the ATP-binding site (PDB code: 6HHO, ). ( J ) RIPK1 reporter signals with indicated mutations (described in G) upon exposure to GSK547 and Necrostatin 1 μM, and the MEKi Cobimetinib (1 μM, control experiment) or DMSO for 1 hr (mean ± SD, n=6 ind. experiments, HEK293T RIPK1 KO). Statistical significance for C–J: one-sample t-test (*p<0.05, **p<0.01, ***p<0.001). Figure 3—source data 1. Raw unedited western blots underlying and shown in . Figure 3—source data 2. Uncropped and labelled western blots and shown in .

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Activation Assay, Over Expression, Expressing, Mutagenesis, Functional Assay, Knock-Out, Binding Assay, Control, Western Blot

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Representative western blots of RIPK1 and RIPK3 kinase conformation (KinCon) expressions after TBZ treatment for indicated time points. HEK293T cells were transfected with KinCon constructs and subjected to bioluminescence measurements after 48 hr. ( B ) 3D structure of RIPK1 with the inhibitor GSK547 (PDB code: 6HHO, ), which binds to an allosteric site in close proximity to ATP. For comparison, the structure of RIPK2 (light gray) in complex with the ATP analogue AMP-PCP (green sticks) is aligned (PDB code: 5NG0, ). ( C ) Time-dependent treatment of RIPK1-K45A KinCon with two RIPK1i (GSK547 and Necrostatin, 1 μM each). The KinCon reporter was over-expressed in HEK293T RIPK1 KO cells for 48 hr. Points represent the mean RLU fold change relative to time point zero for n=3 independent experiments with ± SEM.

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Western Blot, Transfection, Construct, Comparison

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Conformation dynamics of indicated RIPK1 KinCon reporter upon exposure to the two RIPK1i (GSK547 and Necrostatin 1 μM), and the MEKi Cobimetinib (1 μM, control experiment) or DMSO for 1 hr. Bars represent the RLU fold change relative to the DMSO control of wild-type (wt) RIPK1 (mean ± SEM, n=6 ind. experiments, HEK293T RIPK1 KO). ( B ) RIPK1 KinCon auto-phosphorylation with co-expressed Flag-tagged RIPK1 constructs following expression in HEK293T RIPK1 KO cells. Indicated RIPK1 wt or mutant constructs were co-expressed for 48 hr and subjected to western blotting. KinCon RIPK1 constructs are annotated with single asterisk and Flag-tagged RIPK1 with double asterisks. Figure 3—figure supplement 2—source data 1. Raw unedited western blots shown in . Figure 3—figure supplement 2—source data 2. Uncropped and labelled western blots shown in .

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Control, Construct, Expressing, Mutagenesis, Western Blot

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A ) Illustration of regulatory CDK4/6 interactions and Rb activation. ( B ) Domain organization of CDK4, CDK6, and p16 INK4a ; tested point mutations are listed. ( C ) 3D structure of CDK6 in complex with p16 INK4a . Crucial amino acids involved in the interaction of the two proteins are highlighted. The R31C mutant is depicted in orange (PDB code: 1BI7, ). ( D ) Protein:protein interaction (PPI) reporter analyses of the kinases CDK4 and CDK6 with p16 INK4a . Scheme illustrates CDK4/6 hetero-dimer formation with p16 INK4a analyzed using a PCA R Luc PPI reporter system. PPI induces the complementation of R Luc PCA fragments promoting an increase in bioluminescence (HEK293T cells, 48 hr of transient reporter expression). Bars represent the RLU fold change of PPI in relation to wild-type CDK4/6:p16 INK4a complex (mean ± SEM, n=7 ind. experiments). ( E ) Basal signal of CDK4/6 KinCon reporters with indicated mutations are shown (expressed for 48 hr in HEK293T cells) (mean ± SEM, n=6 ind. experiments). ( F ) Quantification of alterations of CDK4/6 KinCon reporter bioluminescence signals (HEK293T, expression for 48 hr) upon exposure to indicated CDK4/6i (1 μM) or DMSO for 3 hr (mean ± SEM, n=4 ind. experiments). Statistical significance for D–F: one-sample t-test (*p<0.05, **p<0.01, ***p<0.001). Figure 4—source data 1. Raw unedited western blots shown in . Figure 4—source data 2. Uncropped and labelled western blots shown in .

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Activation Assay, Mutagenesis, Expressing, Western Blot

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: ( A+B ) Depiction of molecular interactions of a type I 1/2 and type III kinase inhibitors with a kinase domain (N and C lobe). Impact of PLX8394, Cobimetinib and GSK547 on wild-type (wt) and mutated versions of BRAF, RIPK1, and MEK1 kinase conformation (KinCon) reporters. 48 hr post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 1 hr (1 μM) followed by RLuc PCA analyses (mean ± SEM, n=4/5 ind. experiments). ( C ) Depiction of molecular interactions of a type I kinase inhibitor with a kinase domain (N and C lobe). Impact of Abemaciclib on indicated CDK6 kinase conformations (wt and p16 INK4a binding deficient). 48 hr post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 3 hr (1 μM) followed by RLuc PCA analyses (mean ± SEM, n=4 ind. experiments). ( D ) Bioluminescence measurement of PKAc wt and L206R KinCon reporters. HEK293T cells expressing the reporter were treated with 20 μM of Forskolin for 15 min followed by RLuc PCA analyses (mean ± SEM, n=4 ind. experiments). ( E ) Kinome tree displays kinases for which KinCon reporters have been generated (red dots). The blue squares highlight the kinases for which approved drugs are available. Generated with https://kinhub.org/kinmap/ . Statistical significance for A–D: one-sample t-test (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Transfection, Expressing, Construct, Binding Assay, Generated

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet: BRAF and PKAc wild-type (wt) and mutated (BRAF-V600E, PKAc-L206R) reporters were over-expressed in HEK293T cells. ( A ) Representative western blot from the cytoplasmic ( C ) and nuclear (N) fractions. (B+C) Quantifications of the signals from n=3 ind. experiments (mean ± SEM). Figure 5—figure supplement 1—source data 1. Raw unedited western blots shown in . Figure 5—figure supplement 1—source data 2. Uncropped and labelled western blots shown in .

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Western Blot

Journal: eLife

Article Title: Impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/eLife.94755

Figure Lengend Snippet:

Article Snippet: For the HEK293 RIPK1 KO cells HEK293T parental cells were transfected with pMA-T-gRNA-RIP1 targeting (synthetic construct expressing the guide) and Cas9-GFP (pSpCas9(BB)-2A-GFP-Addgene 48138).

Techniques: Transfection, Construct, Luciferase

Journal: bioRxiv

Article Title: Cryo-electron tomography pipeline for plasma membranes

doi: 10.1101/2024.06.27.600657

Figure Lengend Snippet: a, Projection of 21 z-slices from a tomogram of an isolated plasma membrane. 80S ribosomes (black arrows) are frequently found in unroofed HEK293 cells overexpressing dynamin-1(K44A). b, Rotated views (top) and a clipped view (bottom) of a consensus subtomogram average filtered according to the local resolution. c, Fourier shell correlation (FSC) profiles obtained from subtomogram averages. The nominal resolution is reported at FSC=0.143. d,e, Classification of the set of well-aligning particles obtained subtomogram averages of the 80S ribosome in non-rotated and rotated states. tRNA occupying the P, P/E, and A/P sites are indicated in orange, pink, and blue, respectively ( d ) and a subset of 446 membrane-bound ribosomes (two views, e ). f, A view from a tomogram with the top and bottom air-water interfaces (AWIs) indicated (black arrows). g, Distances of putative ribosomes were measured from both AWIs. The number of particles falling within successive 5 nm bins from the bottom and top AWIs (left and right panels, respectively) is plotted for the set of particles obtained immediately following picking (cyan) and the set of well-aligning particles obtained from classification (purple). h, The fraction of particles found with particle picking that constitute the well-aligning class are plotted with respect to their distance to the bottom and top AWIs.

Article Snippet: To generate inducible cells expressing Dynamin1(K44A)-GFP, parental Flp-In T-Rex HEK293 cells (Thermo Fisher, R78007) were co-transfected with a 1:5 mass ratio of pcDNA/TO/Dyn1-K44A-GFP and the Flp recombinase expression plasmid, pOG44 (Thermo Fisher, V600520 ) using Lipofectamine 3000 (Invitrogen, L3000-015) following manufacturer’s instructions.

Techniques: Isolation, Membrane

Journal: bioRxiv

Article Title: Cryo-electron tomography pipeline for plasma membranes

doi: 10.1101/2024.06.27.600657

Figure Lengend Snippet: a, TIRF microscopy of a HEK293 cell expressing FerriTag (FRB-mCherry-FTH1, magenta, and FTL) and Hip1R-GFP-FKBP (green) shown before rapamycin addition (left) and 30-60 seconds after rapamycin addition (right). b, Cryo-fluorescence microscopy of HEK293 isolated plasma membrane on a grid expressing FerriTag (FRB-mCherry-FTH1, magenta, and FTL) and Hip1R-GFP-FKBP (green) with combined colors on the right. c (left), CryoEM montaged map of the same grid square. c(middle), Reflection image acquired on the cryo fluorescence microscope and used to register the images. c(right), Registered fluorescence and EM images combined. Orange dots = membrane outline, arrows = position of d acquisition. d, Tomogram of Hip1R/FerriTag labelling and an example of a prominent clathrin structure (right). Empty FerriTag structures are clearly visible as 12 nm circles (arrow). e, Selected tomogram from HEK293 cells with labeled FerriTag on GFP-FKBP-LCa shown and an example of a well-defined clathrin structure (right). For d and e , a cartoon at the top right depicts the location of the membrane (gray), clathrin (green), and FerriTag (purple). f, A histo-gram of the FerriTag distance from clathrin coated membrane (shown at 0-600 nm and 0-80 nm) for clathrin light chain (GFP-FKBP-LCa) and Hip1R (Hip1R-GFP-FKBP). Molecular models of Ferritin (PDB-1fha), mCherry (PDB-2h5q), FRB/FKBP (PDB-3fap), and GFP (PDB-5wwk) and EM density of the clathrin cage (emdb-21608) at scale with the zoomed-in histogram in combination with cartoons of Hip1R, actin, and membrane are shown as putative models explaining the data to the left. The dashed orange line guides the eye from the data peaks to the center of the FerriTag model. g, An example of FerriTag Hip1R labeling and surrounding a clathrin structure. Segmentation of membrane (gray), clathrin (green), FerriTag (purple) and actin (blue) are shown to the right. h, Close-up examples showing Hip1R density adjacent to FerriTag. Cartoons of membrane (light gray), clathrin (green), Hip1R (dark gray), FerriTag (purple), and actin (blue) are shown to the right to aid image interpretation. Tomogram images in d,e are minimum intensity projections (mIPs) of 21 Gaussian-smoothed XY slices. Tomogram images in g,h are mIPs of 10 Gaussian-smoothed slices. Scale bars are a, 20 μm, 8 μm inset full-width; b,c, 10 μm; d,e, 200 nm, 50 nm inset; g, 100 nm; h, 30 nm.

Article Snippet: To generate inducible cells expressing Dynamin1(K44A)-GFP, parental Flp-In T-Rex HEK293 cells (Thermo Fisher, R78007) were co-transfected with a 1:5 mass ratio of pcDNA/TO/Dyn1-K44A-GFP and the Flp recombinase expression plasmid, pOG44 (Thermo Fisher, V600520 ) using Lipofectamine 3000 (Invitrogen, L3000-015) following manufacturer’s instructions.

Techniques: Microscopy, Expressing, Fluorescence, Isolation, Membrane, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidic Acid Receptor 3 (LPA3): Signaling and Phosphorylation Sites

doi: 10.3390/ijms25126491

Figure Lengend Snippet: LPA 3 receptors expressed are functional and markedly sensitive to heterologous but not to homologous desensitization. Panel ( A ), Representative calcium tracings in response to 1 µM LPA (arrow) in HEK 293 cells (green line) and HEK293 TREx TM transfected to express LPA 3 receptors (induced, black continuous line, and not induced, black dotted line). Panel ( B ), calcium response to 1 µM LPA (arrow) of LPA 3 -expressing cells incubated in buffer with (black line) or without (red dotted line) calcium. Panel ( C ), intracellular calcium tracings of LPA 3 -expressing cells preincubated for 30 min in the absence of any agent (black line) or presence of 10 µM U73122 (phospholipase C inhibitor, orange line) or 10 µM U73343 (inactive analog, red line) and challenged with 1 µM LPA (arrow). Thapsigargin 1 µM (Thap, second arrow). Panel ( D ), calcium tracings of induced cells preincubated overnight without (black line) and with pertussis toxin (purple line); arrow indicates the addition of 1 µM LPA. Panel ( E ), calcium tracings in response to 1 µM LPA of cells preincubated without any agent (black line) or with 1 µM LPA for 5 min and washed before re-challenging (black dotted line) with 1 µM PMA (PMA, red line). Panel ( F ), calcium tracings in response to LPA of cells preincubated without any agent (None, open bar), with 1 µM LPA for 5 min and washed before re-challenging (LPA, dashed bar) or with 1 µM PMA (PMA, red dashed bar). The means are plotted, and vertical lines indicate the SEM of 4–5 experiments performed on different days and distinct cell cultures. *** p < 0.001 vs. baseline.

Article Snippet: Parental Flp-In T-Rex HEK293 cells and the plasmid, pOG44, were obtained from Invitrogen (Carlsbad, CA, USA).

Techniques: Functional Assay, Transfection, Expressing, Incubation